Cucurbitacin B (cucB) is a triterpenoid constituent of Cucurbitaceae vegetables and a promising phytochemical for cancer prevention. However, the mechanism of anti-tumor activity of cucB remains unknown, especially in colon cancers. Here, we demonstrate for the first time that cucB inhibited growth of human colon cancer SW480 cells through a reactive oxygen species (ROS)-dependent mechanism. CucB induced G(2) phase arrest and apoptosis in a dose-dependent manner. At the molecular level, cucB reduced the expression of cyclin B1 and cdc25C proteins and activated caspases in SW480 cells. On the other hand, the state of phosphorylation of signaling transducer and activator of transcription 3 (STAT3) was unchanged. We found that cucB increased intracellular ROS levels, and N-acetylcysteine, a well-known antioxidant, reduced the changes in expression of the molecules, and suppressed both G(2) arrest and apoptosis. These results suggested that cucB induced G(2) arrest and apoptosis through a STAT3-independent but ROS-dependent mechanism in SW480 cells.