Many evidences have shown that dietary intake of cruciferous vegetables could protect against the risk of various types of malignancies. Benzyl isothiocyanate (BITC), one of the compounds from cruciferous vegetables, had shown induced cell cycle arrest and apoptosis in cancer cells. However, there is no available information to address that BITC affects murine leukemia cells in vitro and in vivo. Here, we investigated in vitro effects of BITC on murine leukemia WEHI-3 cells. BITC decreased the percentage of viable cells via G0/G1 arrest and apoptosis in WEHI-3 cells. BITC induced apoptosis through the dysfunction of mitochondria (decreased the levels of mitochondria membrane potential) and activation of caspase-3. Then we investigated in vivo effects of BITC on murine leukemia WEHI-3 cells and the results indicated that BITC decreased the weights of liver and spleen and it also decreased the percentage of CD11b and Mac-3 markers, indicating that the differentiation of the precursor of macrophage and B cells was inhibited. BITC promoted the activity of macrophage phagocytosis in cells which are isolated from PBMC and peritoneal (i.p.). Taken together, BITC can affect WEHI-3 cells in vitro and in vivo.