His laboratory work consists of the Ai-project, which focuses on the language-like skills, number-concepts, and memory ability of a female chimpanzee named Ai.

Matsuzawa has also studied tool use in the wild chimpanzees at Bossou, Guinea, West Africa since 1986.

Bossou chimps are well known to use a pair of stones as hammer and anvil to crack open oil-palm nuts.

Long-term research on wild chimpanzee tool use revealed interesting topics like handedness of use of a hammer, critical period of learning nut-cracking at around 3 to 5 year old, "education by master-apprenticeship" and observational learning, possession of stones, deception, new tool use like algae-scooping, use of leaves for cushions, cultural variation in adjacent communities, etc.

In 2020, Kyoto University announced that Matsuzawa was involved in misappropriating the funding of Primate Research Institute.