Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis due to a combination of late-stage diagnosis and limited response to radiation and chemotherapy. Effective treatments for PDAC are urgently needed. Mutant Kras has been reported as a major cause of glutamine (Gln) addiction in PDAC. Here we investigated the effects of Gln depletion on PDAC cell survival and metabolism. Our data identified Gln as critical for PDAC cell viability. Gln depletion significantly altered choline metabolism in the cell lines investigated.