L. paracaseisubsp.paracaseiX12 was obtained from traditional cheese produced in northwestern China. In this study, we showed that whole peptidoglycan (WPG), extracted fromL. paracaseisubsp.paracaseiX12, inhibited proliferation and induced apoptosis in HT-29 cells in a dose-dependent manner. In addition, WPG-induced apoptosis was associated with the loss of mitochondrial membrane potential (Ψm), the release of cytochrome c (Cyto-C) from mitochondrialto cytosolic spaces, activation of Caspase 3, and accumulation of intracellular reactive oxygen species (ROS). Finally, semi-quantitative RT-PCR showed that these events were accompanied by upregulation of proapoptotic genes (Bax or Bad) and downregulation of antiapoptotic genes (Bcl-xl). Taken together, our results demonstrated that WPG induced apoptosis in HT-29 cells through activation of the mitochondrial pathway. WPG exerted only minor toxicity upon noncancerous cells and therefore might be used as a natural agent in the treatment of cancer in future.