The importance of the absolute configuration of cholesterol for its function in vivo is unknown. To directly test this question in vivo, we synthesized the enantiomer of cholesterol (ent-cholesterol) and tested its ability to substitute for natural cholesterol (nat-cholesterol) in the growth, viability, and behavior of Caenorhabditis elegans, a cholesterol auxotroph. First-generation animals grown on ent-cholesterol were viable with only mild behavioral defects. However, ent-cholesterol produced 100% lethality/arrest of their second generation progeny. Isotopically labeled ent-cholesterol incorporated into animals, indicating that its lethality was not secondary to cholesterol starvation. When mixed with nat-cholesterol, ent-cholesterol was not inert; rather, it antagonized the activity of nat-cholesterol. These results demonstrate for the first time that the absolute configuration of cholesterol, not just its physical properties, is essential for its functions in vivo.