This study demonstrated gemcitabine-induced cytotoxicity in the insulinoma cell line INS-1. Gemcitabine inhibited INS-1 cell proliferation and maintained consistent cell number for 24 h, and then caused apoptosis within 48 h of incubation. Since gemcitabine activates the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which is involved in the resistance of pancreatic exocrine cancer to gemcitabine, we investigated the participation of this pathway in gemcitabine-induced cytotoxicity in INS-1 cells. LY294002 and wortmannin, two PI3-K inhibitors, significantly prevented gemcitabine-induced cytotoxicity in INS-1 cells, indicating that the PI3-K/Akt pathway is involved in gemcitabine-induced cytotoxicity. Gemcitabine-induced Akt phosphorylation in INS-1 cells was prevented by LY294002. Although gemcitabine induced cell cycle arrest at the G1 and early S phases, LY294002 did not inhibit the cell cycle. These data suggest that PI3-K activation does not influence gemcitabine-induced cell cycle arrest. In gemcitabine-treated cells, nuclear fragmentation and DNA ladder formation were observed. These findings suggest that gemcitabine induced apoptotic cell death in INS-1 cells through the activation of the PI3-K/Akt pathway.