Individuals vary in coloration based on age and sex: males are bright metallic blue while females and juveniles are more yellow.

The pupfish consumes nearly every available food resource at Devils Hole, including beetles, snails, algae, and freshwater crustaceans, with diet varying throughout the year.

Efforts to conserve the wild population have included removing sediment from the shallow shelf, adding supplemental food, and installing fences and security cameras to keep unauthorized people away.

[16] These estimates depend heavily on knowledge of the mutation rate in this species, which is unknown, but is predicted to be one of the highest for any vertebrate due to its small population size.

The consumption of the various food resources varies seasonally, though inorganic particulate matter had a high frequency of occurrence in stomach contents year-round in one study.

[23] Predators of the Devils Hole pupfish include the diving beetle species Neoclypeodytes cinctellus, which consumes its eggs and juveniles.

When a disturbance such as an earthquake occurs, it causes the fish to flee en masse into the depths,[21]: 12:58  and begin a spawning event that may be out of season.

[40] Research indicates that the annual population fluctuation is in response to the amount of algae on the shallow shelf, which is dependent on incoming solar radiation and nutrient levels.

Nutrient availability may peak when the cave is used by barn owls as a roosting or nesting site, as their nutrient-rich pellets fall into the water.

The reasons for the second decline are unknown, but inbreeding depression, the loss of a prey species, changing algal and microbial communities, or shifting sediment dynamics have been hypothesized as potential factors.

It could face threats in the future relating to climate change, as warming temperatures in the area are predicted to shorten the period of optimum recruitment, or the time when the next generation is produced and matures.

[42][32][33] The waves caused by earthquakes can scour the algae from the rocks (as well as eggs and larvae), affecting the food supply and spawning grounds.

A 2004 flash flood swept scientific monitoring equipment into Devils Hole, causing the deaths of an estimated eighty pupfish.

[45][46] Isolation over the course of thousands of years has led the Devils Hole pupfish to become what are believed to be one of the most inbred vertebrates on Earth, and the resulting high mutation load and genetic instability remains a potential long-term threat to the species as a whole.

The level continued to drop through 1972, causing alarm as a 1969 study had determined that the shallow shelf was virtually the only feeding and spawning habitat available to the fish.

Conservationists and public opinion began to rally for preservation of the pupfish, with a 1970 issue of the magazine Cry California stating that allowing it to go extinct would be "comparable to bombing the Louvre to make way for a parking lot".

Now, they wanted Spring Meadows to cease using any wells within 4 km (2.5 mi) of Devils Hole for any non-domestic purpose based on the implied reservation doctrine established in Winters v. United States (1908).

[5]: 79  The Court stated that by making Devils Hole part of a National Monument, the groundwater necessary to sustain the pupfish was implicitly reserved.

Fish and Wildlife Service (USFWS) designated about 21,000 acres (8,500 ha) as essential habitat where the groundwater most influenced the water level in the Hole.

The species meets the criteria for this designation due to its extremely small extent of occurrence and area of occupancy, both of which are less than 1 km2 (0.39 sq mi).

[54] After vandalism resulted in the death of a pupfish in 2016, the National Park Service added additional barbed wire to the top of the fences surrounding Devils Hole, also installing more motion sensors and video cameras.

[55] Due to the fear of extinction in the 1960s and 1970s, several measures were taken to create multiple populations of the pupfish outside of Devils Hole to safeguard the species, which is known as ex situ conservation.

Genetic evidence showed that around three individuals of the closely related C. nevadensis, which do have pelvic fins, invaded Point of Rocks between 1997 and 2005, hybridizing with the Devils Hole pupfish.

[58] In May through August 2006, two pupfish from Devils Hole and five from the Hoover Dam Refuge were transferred to a Las Vegas Strip casino aquarium at Mandalay Bay with the hope of understanding how to breed the species in aquaria.

While early breeding efforts appeared successful and four larvae survived to adulthood, all individuals had died by December 2006, possibly from a form of leukemia.

[53] In the early 2010s, a full-scale replica of the upper 6.7 m (22 ft) of Devils Hole was built at the new Ash Meadows Fish Conservation Facility (AMFCF),[60] resulting in a 380,000 L (100,000 U.S. gal) tank.

The temperature is 2–3 °C (3.6–5.4 °F) cooler than that of Devils Hole and the dissolved oxygen content is doubled in attempts to reduce thermal and respiratory stress on the fish.

[62] The legal case over the rights to extract groundwater concluded that the Cappaert family, who invested $7 million into opening a ranch in the area, could no longer withdraw the same amount of water.

[49] The Cappaert family's attorney decried that the Supreme Court had chosen the interests of a fish over people, and a newspaper editor from nearby Pahrump threatened to dump a pesticide into Devils Hole to kill them all.

Increased nutrients for algae growth, swept into Devils Hole by runoff from Hurricane Hilary in 2023, may have contributed to the pupfish's proliferation.