<p>Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal type of cancer with poor diagnosis and prognosis, and overcoming gemcitabine-resistant (Gem-R) is a major obstacle in its treatment. Given the important role of glutamine (Glu) metabolism in tumor drug resistance, we investigated the role and exact mechanism of transglutaminase type 2 (TGM2) in influencing PDAC sensitivity to gemcitabine. In this study, we found that TGM2 exhibited elevated expression levels in Gem-R cells and tissue samples from patients with clinically resistant PDAC. Mechanistically, downregulation of TGM2 suppressed the proliferation of Gem-R PDAC cells both in vitro and in vivo by modulating Glu metabolism. RNA sequencing analysis revealed that the mechanism by which targeting TGM2 inhibits drug resistance in Gem-R PDAC cells may be associated with purinergic receptor P2X7 (P2RX7) within the GO:0014049 pathway (positive regulation of glutamate secretion). P2RX7 is highly expressed in Gem-R PDAC cells and tissue samples, and it participates in Glu metabolism and mitophagy in Gem-R PDAC cells. Furthermore, Glu has also been found to induce mitophagy. Lastly, TGM2 and P2RX7 form a positive feedback regulatory loop, jointly regulating Glu metabolism and mitophagy, thereby promoting drug resistance in Gem-R PDAC cells. These data suggest that the TGM2-P2RX7 loop promotes Gem-R in PDAC by improving Glu metabolism and mitophagy, highlighting its potential as a crucial therapeutic target for PDAC.</p><p></p>