Glutamine is one of the most important metabolites and energy sources for cancer cell proliferation. However, the function of glutamine homeostasis to modulate immunotherapy by regulating immune checkpoint protein expression remains elusive. Here, we report that glutamine abundance increases the expression of the immune checkpoint protein PD-L1 through STAT3 signaling in pancreatic cancer cells. Mechanistically, STAT3 directly interacts with PD-L1 under glutamine stimulation and stabilizes PD-L1 protein by preventing it from ubiquitination and degradation. Additionally, our study suggests that PD-L1 may respond to cellular glutamine levels to regulate mTOR activation and pancreatic cancer cell proliferation. Overall, our study unveils a novel mechanism by which glutamine up-regulates PD-L1 expression to activate mTOR and promote cell proliferation, highlighting a crucial role of PD-L1 in regulating the metabolism of glutamine in pancreatic cancer.