Objective: Chronic myelogenous leukemia (CML) is a genetic disorder in pluripotent stem cell that acquires Philadelphia (Ph) chromosome in 95% of patients. At present, chemotherapy is the most important therapeutic manner for CML patients. Nevertheless, drug resistance and relapse of disease are the main challenges in CML therapy. To overcome this problem, it is necessary to screen new drugs especially from natural sources for cancer therapy. Recently, cytotoxic and anti-cancer activity of microbial secondary metabolites produced by Streptomyces genous has been reported. In this study, we investigated the effects of acetonitrile extract isolated from Streptomyces calvus fermentation broth in K562 cell line as a model of CML. Materials and Methods: K562 cell line was treated by various concentrations of acetonitrile extract (10-1000 ng/ml) for 12, 24, 48 and 72 hours intervals. Anti-proliferative effects of acetonitrile extract were studied by trypan blue exclusion test. Morphological changes in cells were evaluated by light microscope. Results: Acetonitrile extract induced growth inhibition and differentiation of K562 cells in a dose- and time-dependent manner. For example, cell growth decreased 45-53% after 12 to 72 hours. In higher concentrations of the drug (700ng/ml and 1000 ng/ml) and after 72 hours treatment, some of the K562 cells develop a spindle-shaped morphology and showed other morphological changes such as pseudopod formation suggesting differentiated K562 cells. Conclusion: According to growth inhibitory and differentiation effects of acetonitrile soluble metabolite of Streptomyces calvus, this compound maybe a good candidate for treatment of CML patients in differentiation therapy approach.