Selenite has emerged as an optional chemotherapeutic agent for hematological malignancies. Autophagy and apoptosis are both engaged in selenite-induced cell death. In a previous report, we have identified heat shock protein 90 (Hsp90) as a critical modulator of the balance between autophagy and apoptosis in selenite-treated leukemia cells. However, the mechanisms by which selenite mediates the crosstalk between autophagy and apoptosis remain largely unknown. Herein, we demonstrate that the endoplasmic reticulum (ER) stress-related PERK/eIF2α/ATF4 pathway and p38 are core modules for the selenite-induced switch to apoptosis from autophagy. We found that selenite activated PERK and eIF2α/ATF4 downstream to promote apoptosis. During this progression, p38 was dissociated from PERK-inhibiting Hsp90 and became autophosphorylated. Then, activated p38 further enhanced the docking of activating transcription factor 4 (ATF4) onto the CHOP (CCAAT/enhancer-binding protein homologous protein) promoter via eIF2α to enhance apoptosis. We also found that activated p38 suppressed the phosphorylation of eIF4E that directed ATF4 to bind to the MAP1LC3B (microtubule-associated protein 1 light chain 3B) promoter. Because of the deactivation of eIF4E, the association of ATF4 with the MAP1LC3B promoter was inhibited, and autophagy was compromised. Intriguingly, p53 played important roles in mediating the p38-mediated regulation of eIF2α and eIF4E. When activated by p38, p53 induced the phosphorylation of eIF2α and the dephosphorylation of eIF4E, particularly in the nucleus where the ATF4 transcription factor was modulated, ultimately resulting in differential expression of CHOP and LC3. Moreover, selenite exhibited potent antitumor effects in vivo. In an NB4 cell xenograft model, selenite induced apoptosis and hampered autophagy. In addition, related signaling proteins demonstrated similar changes to those observed in vitro. These data suggest that selenite may be a candidate drug for leukemia therapy.