Sphingolipid metabolites inducing ceramide, sphingosine, and sphingosine-1-phosphate (S1P) play important roles in the regulation of cell proliferation, survival, and death. Aminoglycoside antibiotics including gentamicin induce inner ear hair cell loss and sensorineural hearing loss. Apoptotic cell death is considered to play a key role in this injury. The present study was designed to investigate the possible involvement of ceramide and S1P in hair cell death due to gentamicin. In addition, the effects of other metabolites of ceramide, gangliosides GM1 (GM1) and GM3 (GM3), on gentamicin ototoxicity were also investigated. Basal turn organ of Corti explants from p3 to p5 rats were maintained in tissue culture and exposed to 20 or 35 μM gentamicin for 48 h. The effects of ceramide, S1P, GM1, and GM3 on gentamicin-induced hair cell loss were examined. Gentamicin-induced hair cell loss was increased by ceramide but was decreased by S1P. GM1 and GM3 exhibited protective effects against gentamicin-induced hair cell death at the limited concentrations. These results indicate that ceramide enhances gentamicin ototoxicity by promoting apoptotic hair cell death, and that S1P, GM1, and GM3 act as cochlear protectants. In conclusion, sphingolipid metabolites influence the apoptotic reaction of hair cells to gentamicin ototoxicity.