3-Hydroxysteroid-24 reductase (DHCR24) is an endoplasmic reticulum (ER)-resident, multifunctional enzyme that possesses anti-apoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H₂O₂) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24^-/- MEFs). H₂O₂ exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase (ASK)-1 and stress-activated protein kinases, such as p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Complementation of the MEFs by adenovirus expressing DHCR24 attenuated the H₂O₂-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H₂O₂ was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such anti-apoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced ER stress by eliminating ROS. Finally, utilizing in vitro synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H₂O₂-scavenging activity, while the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H₂O₂, thereby protecting cells from oxidative-stress-induced apoptosis.