Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a median 5 year-survival rate of 8%. Cannabidiol (CBD) has been found to exhibit antineoplastic potential and may potentiate the anticancer effects of gemcitabine. The biological mechanism for this synergy remains elusive but in other tumour types, CBD therapy has been linked to de novo synthesis of ceramide. The sphingolipid ceramide is a potent tumour suppressor lipid with roles in apoptosis and autophagy. The aim of this study was to establish an in vitro platform to investigate the cytotoxic mechanism of action of CBD in PDAC, evaluate the potential synergy with gemcitabine therapy, and describe the effect on ceramide induction in this pathway. The findings point to evidence of a putative ceramide synthase 1 (CerS1) dependent pathway driven by CBD in activating endoplasmic reticulum (ER) stress target; GRP78. Specifically, in gemcitabine resistant cells, Panc1, the activation of the GRP78/ATF4/CHOP arm of the unfolded protein response (UPR) pathway was observed. In gemcitabine sensitive cells, Panc03.27, CerS1 showed enhanced upregulation of CerS1, GRP78, CHOP targets in combination with gemcitabine. The involvement of a cannabinoid receptor was analysed by viability and protein expression which showed evidence of CB2 and GPR55 expression in both human and murine cells. Cell cycle analysis showed arrest in G0-G1 phase and an increase in apoptotic cells via annexin/PI studies, indicating cell death specific effects of CBD in pancreatic cancer cells. Using an in vivo model of PDAC, we aimed to characterise if CBD could sensitise cells to gemcitabine and abraxane. No significance in survival of animals nor changes to immune populations of processed tumours were observed. However, double chemotherapy combination with co administration of CBD showed a significant reduction in tumour burden. These findings provide evidence of a cytotoxic mechanism of action of CBD in pancreatic cancer.