Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.