ARTICLE ABSTRACTSenescence, an inherent tumor suppressive mechanism, is a critical determinant for chemotherapy. In the present study, we show that the monocarboxylate transporter 2 (MCT2) protein was tumor-selectively expressed in human colorectal malignancies and knockdown of MCT2 induces mitochondrial dysfunction, cell-cycle arrest, and senescence without additional cellular stress in colorectal cancer cell lines. Moreover, the reactive oxygen species (ROS) scavenger, N-acetylcysteine, blocked MCT2 knockdown-induced growth arrest and cellular senescence, indicating a pivotal role of ROS in this pathway. Dramatic induction of mitochondrial superoxide generation and decrease in ATP production was observed, indicating that mitochondrial dysfunction is the major mechanism underlying MCT2 knockdown-induced ROS generation. Senescence-associated DNA damage was also evident from the increase in promyelocytic leukemia bodies, γH2AX foci, and SAHF. Conversely, overexpression of MCT2 prevented doxorubicin-induced ROS accumulation (P = 0.0002) and cell growth inhibition (P = 0.001). MCT2 knockdown suppressed KRAS mutant colorectal tumor growth in vivo. In addition, MCT2 knockdown and cytostatic drug combination further enhanced the antitumor effect. These findings support the use of MCT2 as a promising target for inhibition of colorectal cancer. Mol Cancer Ther; 11(11); 2342–51. ©2012 AACR.