Abstract Cytotoxic T Lymphocytes (CTLs) are the major effectors of the host cancer immune surveillance. FasL-induced cytotoxicity is one of the two effector mechanisms that CTLs use to kill tumor cells. Fas is the physiological ligand of FasL and its expression and function is often deregulated in cancer cells. Ceramide is a sphingolipid metabolite that mediates Fas function. We aimed at testing the hypothesis that ceramide analogs are effective in modulating Fas function to sensitize colon carcinoma cells to FasL-induced apoptosis by tumor-specific CTLs. We show that Fas is expressed in human colon carcinoma cells. However, human colon carcinoma cells are not sensitive to FasL-induced apoptosis. Based on structures of existing ceramide analogs and ceramidase inhibitors, we used rationale design and synthesized twenty ceramide analogs as Fas function modulators. Six of these twenty ceramide analogs, IG4, IG7, IG8, IG14, IG17 and IG19, exhibit potent activity in sensitization of human colon carcinoma cells to FasL-induced apoptosis. At the molecular level, we observed that these six ceramide analogs dramatically increased FasL-induced activation of caspase 8, an essential initiator caspase of the Fas receptor-death-inducing signaling complex. Finally, a sublethal dose of ceramide analog significantly increased CTL-mediated and FasL-induced apoptosis of colon carcinoma cells. We have therefore developed six novel ceramide analogs that act at sublethal doses to enhance the efficacy of tumor-specific CTLs and hold great promise for further development as adjunct agents in CTL-based colon cancer immunotherapy.