Background : Although royal jelly (RJ) have been shown to promote wound healing by increasing sphingolipids, the mechanism have not been elucidated. Object: We investigated the effect of RJ on the content sphingoid base which is the basic component of sphingolipid, a major lipid of epidermal barrier, protein and mRNA expressions of relative enzyme. Methods: The scratched-wounded human dermal fibroblasts were treated with royal jelly (0.1,1,5μg/ml) for up to 8hrs. The contents of sphingoid base were analyzed by HPLC. The protein and mRNA expression of ceramide synthase and acid ceramidase were analyzed by western blotting and real time PCR. Result: The amount of sphingoid base such as C17-So, C18-So, C18-Sa in the all of RJ treated groups were significantly decreased compared to control group. Protein expressions of ceramide synthase in RJ treated group were significantly increased compared to control group. But the protein expression of acid ceramidase was not altered. Furthermore mRNA expression of ceramid e synthase and acid ceramidase did not differ in all groups. Conclusion: RJ decrease the content of sphingoid base coupled with increased protein expression of ceramide synthase in an in vitro scratched-wound human dermal fibroblasts.