As a potential novel agent for treating pancreatic cancer, methylseleninic acid (MSeA) was evaluated in cell culture and xenograft models. Results showed that MSeA induced G1 cell cycle arrest and apoptosis in a majority of human and mouse pancreatic cancer cell lines, but G2 arrest in human PANC-1 and PANC-28 cell lines. In contrast to our previous finding in human prostate cancer LNCaP cells having a lack of P53 activation by MSeA, induction of G2 arrest in PANC-1 cells was accompanied by increased mutant P53 Ser15 phosphorylation, upregulation of P53-targets P21Cip1 and GADD45 and G 2 checkpoint kinase (Chk2) activation, suggestive of DNA damage responses. A rapid inhibition of AKT phosphorylation was followed by reduced mTOR signaling and increased autophagy in PANC-1 cells attenuating caspase-mediated apoptosis execution. Furthermore, daily oral treatment with MSeA (3 mg Se/kg body weight) significantly suppressed growth of subcutaneously inoculated PANC-1 xenograft in SCID mice. Immunohistochemical analyses detected increased p-Ser15 P53, P21Cip1, pS139-H2AX (DNA damage responses), and caspase-3 cleavage and decreased pSer473AKT and Ki67 proliferative index and reduced intratumor vascular density in MSeA-treated xenograft. These results provide impetus for further research of MSeA in the therapy and/or chemoprevention of pancreatic cancer.