The Lactobacillus paracasei subsp. paracasei M5 strain exerted potential anticancer activity through the cell wall. In this study, whole peptidoglycan (WPG) was extracted from the Lactobacillus paracasei subsp. paracasei M5 strain and was evaluated for anticancer effects as well as its properties. SDS-PAGE analysis confirmed the presence of WPG with dominant bands of approximately 14.4 kDa. Further analysis revealed that the amino acids present in the WPG consisted of alanine, glycine, glutamic acid, and lysine in a molar ratio of approximately 8 : 5 : 3 : 3.5. In addition, the cell viability of HT-29 cells with WPG addition was investigated with methyl thiazolyl tetrazolium (MTT) and trypan blue exclusion (TBE) assays, and cell apoptosis was analyzed with a transmission electron microscope, flow cytometry, and semiquantitative RT-PCR. These results showed that WPG exerted cytotoxic effects on colon cancer HT-29 cells in a dose-dependent manner and upregulated proapoptotic genes, while downregulating antiapoptotic genes. The gene expression study definitively revealed that WPG induced a mitochondria-mediated apoptotic pathway.