Abstract Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor, which is abnormally expressed in myeloid leukemia and other several solid cancers. It is associated with short survival as well as anticancer drug resistance. Autophagy is a protective mechanism that promotes cancer cell growth and survival under stressed conditions including clinical drug treatment. Here evidences are provided that EVI1 induces autophagy and mediated drug resistance in myeloid leukemia cells. Both knockdown using RNAi and pharmacological inhibition of autophagy significantly increase sensitivity to cytotoxic drug treatment in EVI1high cells. Mechanistic studies revealed that EVI1 regulated autophagy by directly binding to autophagy-related gene autophagy related 7 (ATG7) promoter and transcriptionally upregulating its expression. Notably, ATG7 expression was positively correlated with EVI1 in bone marrow mononuclear cells from myeloid leukemia patients. Acute myeloid leukemia patients with high level of EVI1 are associated with unfavorable overall survival, which was aggravated by simultaneous high expression of ATG7 in these patients. Furthermore, ChIP and firefly luciferase reporter assay identified an EVI1-binding site at 227 upstream promoter region of ATG7 which regulated its transcription. In addition, enforced expression of EVI1 also increased intracellular reactive oxygen species and ATG7 mRNA levels as well as autophagy activity, whereas the increase was attenuated after treatment with reactive oxygen species scavenger, suggesting the involvement of reactive oxygen species in EVI1-induced autophagy. These findings demonstrate that EVI protects myeloid leukemia cell from anticancer drug treatment by inducing autophagy through dual control of ATG7. These results might present a new therapeutic approach for improving treatment outcome in myelogenous leukemia with EVI1high.