OBJECTIVES: Increased expression of glutaminase (GLS) has been found to correlate with more aggressive disease and poorer prognosis in patients with several types of cancer, including breast, lung, and pancreatic cancer. G9a histone methyltransferase inhibitors may have anticancer activity. The present study assessed whether BIX01294 (BIX), a G9a histone methyltransferase inhibitor, can inhibit glutaminase (GLS) in pancreatic ductal adenocarcinoma (PDAC) cells. METHODS: The effects of BIX on mitochondrial metabolism in PDAC cells were evaluated by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomic analysis. To assess the impact of BIX on glutathione dynamics, real-time changes in glutathione levels were monitored by FreSHtracer-based GSH assays. RESULTS: BIX significantly inhibited the growth of PDAC cells, both in vitro and in vivo, and robustly induced apoptotic cell death. BIX significantly increased the cellular NADP CONCLUSIONS: Collectively, these findings suggest that BIX could be a potent therapeutic agent in patients with PDAC through its inhibition of GLS-mediated cellular redox balance.