This study investigated the effect of cyclopamine, a hedgehog signaling inhibitor, on immune-related gene expression in rainbow trout head kidney leukocytes in vitro. At 1h sampling time COX 2, IFN-2, TNF-alpha, C3, MHC-II, Il-1 beta, IL-12, IL-10, and IL-6 increased significantly in groups exposed to cyclopamine at 30 mu g/ml. However, in groups exposed to 10 and 20 mu g/ml cyclopamine, the expression of these genes decreased significantly. At 4h sampling time, levels of COX-2, IFN-1, IFN-2, TNF-alpha, TGF-beta, IgT, and MHC-II in groups exposed to 20 mu g/ml cyclopamine increased. At 8h sampling time, COX2, IFN-Reg, and TNF-alpha expression in groups exposed to 10 and 30 mu g/ml cyclopamine increased significantly compared to those in the controls. Conversely the expression decreased significantly for almost all other genes. At 12h sampling time, almost all genes increased significantly in groups exposed to 10 mu g/ml cyclopamine compared to those in the controls. Notably, after 24h, the gene expression in all groups significantly decreased compared to the controls. Our results suggest that the activation of cyclopamine can be a useful tool for the examination of immune-related gene expression in the rainbow trout.