Human carcinomas often show resistance to cisplatin and Bcl-2 is associated with resistance to cisplatin. However, Bcl-2 regulation on cisplatin treatment in human cancers is unknown. Here, we show a novel mechanism by which cisplatin treatment promotes resistance by increasing the expression of Bcl-2 mRNA. Bcl-2 mRNA and protein expression was increased in cisplatin-resistant endometrial cancer cell lines (KLE and HEC-1-A), but not in cisplatin-sensitive cell line (Ishikawa). Cisplatin-mediated increase in Bcl-2 expression was blocked by combination with either actinomycin D or cycloheximide. In addition, Bcl-2 inhibition by HA14-1 led to increased cisplatin-induced apoptosis in KLE and HEC-1-A, whereas Bcl-2 overexpression in Ishikawa led to decreased cisplatin-induced apoptosis. Inhibition of protein kinase C (PKC) activity prevented cisplatin-dependant increase in Bcl-2 mRNA, and induced apoptosis in KLE cells. Furthermore, PKC inhibition was associated with decreased Akt and NF-κB activity. Cells stably expressing shRNA for Akt isoforms revealed that Akt2 was involved in cisplatin-dependant increase in Bcl-2 and apoptosis. Overexpression of Akt2 in Akt2-deficient cells led to increased Bcl-2 expression on cisplatin treatment. Our data suggest a novel regulation pathway of Bcl-2 by cisplatin, via the activation of PKC and Akt2, which has a profound impact on resistance to cisplatin-induced apoptosis in endometrial cancer cells.