Cisplatin is a conventional chemotherapeutic agent that binds covalently to purine DNA bases and mediates cellular apoptosis. A better understanding of the downstream cellular targets of cisplatin will provide information on its mechanism of action and help to understand the mechanism of drug resistance. In this study, we have investigated the effects of cisplatin in a panel of colon carcinoma cell lines and the involvement of the phosphoinositide-3-kinase/forkhead/winged helix box class O (FOXO) pathway in cisplatin action and resistance. Cisplatin-sensitive and cisplatin-resistant cell lines have been characterized in cell viability, flow cytometry, and clonogenic assays. The main components of the phosphoinositide-3-kinase/protein kinase B pathway, particularly FOXO3a, have been analyzed in sensitive and resistant cells on cisplatin treatment. Interestingly, in sensitive cells, cisplatin induces FOXO3a dephosphorylation and nuclear translocation, and expression of its target genes, whereas in resistant cells the effect of cisplatin on FOXO3a is incomplete. Consistent with this, protein kinase B/FOXO signaling axis modulators triciribine and psammaplysene A sensitize the resistant HT29 cells to cisplatin treatment. Critically, knockdown of FOXO3a expression using small interfering RNA rescues sensitive SW620 cells from cisplatin-induced short- and long-term cell death. Together, our findings suggest that FOXO3a is a relevant mediator of the cytotoxic effects of cisplatin in colon cancer cells.