Pancreatic cancer, the fourth most common cause of cancer-related deaths, is one of the most aggressive and devastating human malignancies with increasing incidence worldwide. To date, surgical resection is the only potentially curative therapy available for pancreatic cancer patients. Early diagnosis of pancreatic tumors is difficult, and hence, nearly 80% of patients cannot receive surgical resection. Natural products have always been a vital source for novel compounds for cancer treatment. The naturally occurring prenylated xanthone, gambogic acid, has been previously shown to exert potent anticancer, anti-inflammatory, apoptotic, antiangiogenic, and antioxidant activities. However, to our knowledge, there have been no specific studies showing its effect on the whole-genome expression in pancreatic cancer cells. Here, the anticancer activity of gambogic acid toward a panel of pancreatic cancer cells with different differentiation stages has been evaluated. Additionally, a whole-genome transcription profiling study was performed in order to identify possible candidate players modulating the antitumor effect of gambogic acid on pancreatic cancer cells. Expression analysis results showed that the pancreatic adenocarcinoma signaling pathway was specifically affected upon gambogic acid treatment. Moreover, the growth inhibitory effect of gambogic acid on pancreatic cancer cells was modulated through up-regulation of DDIT3, DUSP1, and DUSP5 and down-regulation of ALDOA, TOP2A, and ATG4B. The present work is a starting point for the generation of hypotheses on significantly regulated candidate key player genes and for a detailed dissection of the potential role of each individual gene for the activity of gambogic acid on pancreatic cancer.