Cucurbitacin B (Cuc B) is natural triterpenoid with potent anti-cancer activities both in vitro and in vivo. This study investigated the anti-cancer effect of Cuc B on K562 leukemia cells. We demonstrated that Cuc B dramatically decreased cell viability in a dose-dependent manner. Cuc B treatment caused DNA damage as evidenced by long tails in comet assay and increased protein expression of γH2AX. Cuc B treatment caused nuclear γH2AX translocation and depolarization of mitochondrial membrane potential (MMP) as determined by immunofluorescence and JC-1 staining respectively. Cuc B induced G2/M cell phase arrest and apoptosis as determined by flow cytometry, DNA fragmentations, and protein expressions. In addition, Cuc B dramatically increased intracellular reactive oxygen species (ROS) formation as measured by DCFH2-DA. N-acetyl-l-cysteine (NAC) pretreatment significantly inhibited Cuc B induced DNA damage, G2/M phase arrest, and apoptosis. Taken together, these results suggested that ROS mediated Cuc B-induced DNA damage, G2/M arrest, and apoptosis in K562 cells. This study provides novel mechanisms and potential targets for better understanding the anti-cancer mechanisms of Cuc B.