Tetrandrine is a broadly used bisbenzylisoquinoline alkaloid component of traditional Chinese medicine that has antitumor effects in some cancer types. In this study, we investigated the effects of tetrandrine on leukemia in vitro and in vivo. The results showed that tetrandrine effectively induced differentiation and autophagy in leukemia cells. In addition, tetrandrine treatment activated the accumulation of reactive oxygen species (ROS) and inhibited c-MYC protein expression. Further, we found that treatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and Tiron as well as overexpression of c-MYC reduced tetrandrine-induced autophagy and differentiation. Moreover, a small molecular c-MYC inhibitor, 10058-F4, enhanced the tetrandrine-induced differentiation of leukemia cells. These results suggest that ROS generation and c-MYC suppression play important roles in tetrandrine-induced autophagy and differentiation, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our data suggest that tetrandrine may be a promising agent for the treatment of leukemia.