Ecotropic viral integration site-1 (EVI-1) has been recognized as one of the dominant oncogenes associated with murine and human myeloid leukemia. Recent clinical studies demonstrated that high EVI-1 expression was an independent negative prognostic indicator of survival in leukemia patients. In addition, gene-targeting studies in mice reveal that Evi-1 is preferentially expressed in hematopoietic stem cells (HSCs) and plays an essential role in proliferation/maintenance of HSCs. Proteins associated with EVI-1, signaling pathways regulated by EVI-1, and downstream mediators of EVI-1 transcriptional regulation have been described and characterized. In this study, we summarize current knowledge regarding biochemical properties and biological functions of EVI-1, which provides a foundation for the development of novel therapeutic strategies.