This study investigated the interaction between the novel ceramide analog AL6 and gemcitabine in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, harboring different polymorphic variants of the gemcitabine catabolism enzyme cytidine deaminase (CDA). AL6 dose-dependently inhibited cell growth, induced apoptosis and synergistically enhanced the cytotoxic activity of gemcitabine. Moreover, it triggered apoptosis, which was significantly enhanced by the combination, and increased the ratio between gene expression of the activating enzyme deoxycytidine kinase (dCK) and CDA, potentially favoring gemcitabine activity. In conclusion, AL6 displays synergistic cytotoxic activity, enhances apoptosis, and favorably modulates enzymes involved in gemcitabine metabolism, supporting future investigation of this combination in pancreatic cancer.