Quinacrine, which is clinically used as an antimalarial drug, has anti-cancer activity. However, mechanism underlying its cytotoxic effect remains to be completely elucidated. In the present study, we investigated the cytotoxic effect of quinacrine on human leukemia U937 cells. Quinacrine-induced apoptosis of U937 cells was accompanied with ROS generation, mitochondrial depolarization, and BAX upregulation. Quinacrine-treated U937 cells showed ROS-mediated p38 MAPK activation and ERK inactivation, which in turn upregulated FOXP3 transcription. FOXP3-mediated miR-183 expression decreased β-TrCP mRNA stability and suppressed β-TrCP-mediated SP1 degradation, thus increasing SP1 expression in U937 cells. Upregulated SP1 expression further increased BAX expression. BAX knock-down attenuated quinacrine-induced mitochondrial depolarization and increased the viability of quinacrine-treated cells. Together, our data indicate that quinacrine-induced apoptosis of U937 cells is mediated by mitochondrial alterations triggered by FOXP3/miR-183/β-TrCP/SP1 axis-mediated BAX upregulation.