Cycloheximide exerts its effects by interfering with the translocation step in protein synthesis (movement of two tRNA molecules and mRNA in relation to the ribosome), thus blocking eukaryotic translational elongation.

Cycloheximide is widely used in biomedical research to inhibit protein synthesis in eukaryotic cells studied in vitro (i.e. outside of organisms).

[1] Due to significant toxic side effects, including DNA damage, teratogenesis, and other reproductive effects (including birth defects and toxicity to sperm[2]), cycloheximide is generally used only in in vitro research applications, and is not suitable for human use as a therapeutic compound.

Because cycloheximide rapidly breaks down in a basic environment, decontamination of work surfaces and containers can be achieved by washing with a non-harmful alkali solution such as soapy water or aqueous sodium bicarbonate.

It is also used in media to detect unwanted bacteria in beer fermentation by suppressing yeasts and molds growth in test medium.