BACKGROUND AND AIMS: Elevated PD-L1 expression in tumor cells facilitates immune evasion. However, the mechanism via which PD-L1 expression is regulated in pancreatic ductal adenocarcinoma (PDAC) cells remains inadequately elucidated. METHODS: Immunoprecipitation, pull-down assays, and mass spectrometry were used to identify glutamine synthase 2 (GLS2) and yes1 associated transcriptional regulator (YAP1) binding proteins and modification sites. Immunoblotting, immunofluorescence, chromatin immunoprecipitation, and luciferase reporter assays were used to analyze YAP1 activation. Protein expression levels were assessed using immunoblotting, immunoprecipitation, immunofluorescence, and immunohistochemistry. RNA expression levels were analyzed using RT-qPCR. RESULTS: Hypoxia-induced GCN5-mediated acetylation of GLS2 at K151, which enhanced GLS2 interaction with YAP1. Subsequently, tubulin tyrosine ligase-like 1 mediated YAP1 glutamylation at E100 and promoted its nuclear translocation and the activation-dependent transcriptional up-regulation of PD-L1 expression. The expression of GLS2-K151R or YAP1-E100A mutants in PDAC cells blocked hypoxia-induced PD-L1 expression and enhanced CD4 CONCLUSIONS: The present study revealed a novel mechanism by which hypoxia up-regulates PD-L1 expression and highlighted the involvement of GLS2 in noncanonical metabolic pathways involved in tumor immune evasion, with implications for PDAC treatment.