Indirubin is the active component of Dang gui Long hui Wan, a traditional Chinese herbal medicine used as therapy for chronic myelogenous leukemia (CML). In clinical studies, indirubin seldom caused major side-effects. However, the functional effect of indirubin on acute lymphoblastic leukemia (ALL) is unclear. Therefore, we investigated the effects of indirubin-3'-monoxime (I3M) on the ALL cell line JM1 and the CML cell line K562 (control). The anti-leukemia effects and mechanisms of I3M were similar on ALL and CML cells. I3M significantly and dose-dependently decreased cell viability. The G2/M cell cycle phase was arrested and the sub-G1 proportion was relatively increased. In addition, caspase-3 activation led to poly(ADP-ribose) polymerase (PARP)-1 cleavage and the progression of apoptosis. Notably, I3M induced autophagy. However, I3M had no effect on necrosis in either cell line. We specifically found that I3M only marginally affected the survival of primary mature lymphocytes, and was not cytotoxic to granulocytes. Since I3M induced apoptosis and autophagy in human lymphocytic leukemia cells and caused few side-effects in healthy lymphocytes and granulocytes, I3M may be useful for clinical anti-ALL therapy.