Pharmacological ascorbate induces autophagy in pancreatic cancer, which is characterized by accumulation of autophagosomes and the processing of microtubule-associated protein light chain 3 (LC3) to the lipidated form, LC3- II. Prior studies have demonstrated LC3-II upregulation in pancreatic cancer cells after gemcitabine chemotherapy or ionizing radiation. The aim of this study was to determine the role of autophagy in pharmacological ascorbate-induced cytotoxicity. Pancreatic cancer cells were generated to stably overexpressLC3 fused to a green fluorescent protein (MIA PaCa-2 LC3-GFP). MIA PaCa-2 LC3-GFP cells had a higher LC3-II/LC3-I ratio at 6 and 24 hours and demonstrated a clonogenic survival advantage after pharmacological ascorbate treatment compared to parental cells. In cells with knockdown of Atg5, there was increased susceptibility to pharmacological ascorbate-induced cytotoxicity. Our data indicate autophagy may be an important survival mechanism after pharmacological ascorbate-induced cytotoxicity, while impairment of autophagy can re-sensitize cells to ascorbate. LC3 over expression has a protective effect on cell survival after ascorbate treatment. Autophagy-modulating drugs may be an important adjunct for enhancement of ascorbate-induced cytotoxicity as well as a potential therapy for re-sensitization of chemoradiation-resistant tumors.