Mounting evidence shows that selenium possesses chemotherapeutic potential against tumor cells, including leukemia, prostate cancer and colorectal cancer (CRC) cells. However, the detailed mechanism by which sodium selenite specifically kills tumor cells remains unclear. Herein, we demonstrated that supranutritional doses of selenite-induced apoptosis in CRC cells through reactive oxygen species (ROS)-dependent modulation of the PI3K/AKT/FoxO3a signaling pathway. First, we found that selenite treatment in HCT116 and SW480 CRC cells caused inhibition of AKT and the nuclear accumulation of FoxO3a by western blot and immunofluorescence analyses, respectively, thereby facilitating transcription of the target genes bim and PTEN. Modulation of the AKT/FoxO3a/Bim signaling pathway by chemical inhibitors or RNA interference revealed that these events were critical for selenite-induced apoptosis in CRC cells. Additionally, we discovered that FoxO3a-mediated upregulation of PTEN exerted a further inhibitory effect on the AKT survival pathway. We also corroborated our findings in vivo by performing immunohistochemistry experiments. In summary, our results show that selenite could induce ROS-dependent FoxO3a-mediated apoptosis in CRC cells and xenograft tumors through PTEN-mediated inhibition of the PI3K/AKT survival axis. These results help to elucidate the molecular mechanisms underlying selenite-induced cell death in tumor cells and provide a theoretical basis for translational applications of selenium.