Metformin and aspirin have been studied extensively as cancer preventative and therapeutic agents. However, the underlying molecular mechanisms for the inhibitory effects of pancreatic cancer development remain undefined. To gain further insight into their biological function in pancreatic cancer, we conducted a transcriptomic analysis using RNA sequencing to assess the differential gene expression induced by metformin (5 mM) and aspirin (2 mM), alone or in combination, after treatment of PANC-1 cells for 48 hours. Compared to an untreated control, metformin down-regulated 58 genes and up-regulated 91 genes, aspirin down-regulated 12 genes only, while metformin plus aspirin down-regulated 656 genes and up-regulated 449 genes (fold-change > 2, P < 10(-5)). Of the top 10 genes (fold-change > 10, P < 10(-10)) regulated by metformin plus aspirin, PCDH18, CCL2, RASL11A, FAM111B and BMP5 were down-regulated ≥ 20-fold, while NGFR, NPTX1, C7orf57, MRPL23AS1 and UNC5B were up-regulated ≥ 10-fold. Ingenuity Pathway Analysis (IPA) revealed that the pathways, "cholesterol biosynthesis", "cell cycle: G1/S checkpoint regulation", and "axonal guidance signaling" were the most statistically significant pathways modulated by metformin plus aspirin. Although the results need further functional validation, these data provide the first evidence for the synergistic action between metformin and aspirin in modulating the transcriptional profile of pancreatic cancer cells.