Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme catabolism, is known to modulate various cellular functions, including cytokine production, cell proliferation, and apoptosis, in stress-related conditions. However, the role of HO-1 in the auditory system remains elusive. Herein, we demonstrate that pharmacologic induction of HO-1 along with catalytic activation significantly suppressed apoptosis of HEI-OC1 cells induced by cisplatin. Studies of ectopic expression of pcDNA3-HO-1 and siRNA of HO-1 further revealed the protective role of HO-1 against cisplatin in HEI-OC1 cells. Among the catabolic metabolites of HO-1, both carbon monoxide (CO) and bilirubin were directly involved in the protective role of HO-1 against cisplatin through inhibition of reactive oxygen species generation. Furthermore, pharmacological induction of HO-1 completely prevented the destruction of outer hair cell arrays by cisplatin through a CO-dependent mechanism in organotrophic culture of the rat primary organ of Corti explants. These results suggest that HO-1 may serve as a safeguard of auditory sensory hair cells against a variety of challenges of oxidative stress, including noise trauma, presbycusis, and ototoxic drugs, respectively.