Probiotics are known to be a potential agent for colorectal cancer (CRC) inhibition, but the precise mechanisms by which probiotic exert anti-tumorigenic effects remain to be explored. Lactobacillus salivarius (LS) Ren was isolated from centenarians living in Bama of China, which showed an anticancer potent in animal model of oral cancer. Here, we investigated the effect of LS on colorectal carcinogenesis and its putative mechanism. Oral administration of LS effectively suppressed the formation of dimethylhydrazine (DMH)-induced CRC in both initial and post-initial stages. Significant antiproliferation and proapoptotic effects were observed with inhibition of tumor formation by dietary intake of LS. Besides, LS metabolites inhibited growth, arrested cell cycle, and induced apoptosis of HT-29 cells. Furthermore, upon the treatment of LS, protein kinase B (AKT) phosphorylation and the downstream proteins of cyclinD1 and cyclooxygenase-2 (COX-2) were significantly downregulated in both in vivo and in vitro tests. These results showed that LS inhibited the colorectal carcinogenesis through suppressing AKT signaling pathway, resulting in suppressing cell proliferation and inducing cell apoptosis. Our fmdings suggest that this probiotic may act as a prophylactic agent for CRC prevention.