Streptococcus thermophilus was identified to be depleted in patients with colorectal cancer (CRC) by shotgun metagenomic sequencing of 526 multi-cohort fecal samples. Here, we aim to investigate if this bacterium could act as a prophylactics for CRC prevention.The anti-tumor effects of S. thermophilus were assessed in cultured colonic epithelial cells and in two murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S. thermophilus was identified by mass spectrometry and followed by β-galactosidase activity assay. The mutant strain of S. thermophilus was constructed by homologous recombination. The effect of S. thermophilus on the gut microbiota composition was assessed by shot-gun metagenomic sequencing.Oral gavage of S. thermophilus significantly reduced tumor formation in both Apc and azoxymethane-injected mice. Co-incubation with S. thermophilus or its conditioned medium decreased the proliferation of cultured CRC cells. β-galactosidase was identified as the critical protein produced by S. thermophilus by mass-spectrometry screening and β-galactosidase activity assay. β-galactosidase secreted by S. thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S. thermophilus without functional β-galactosidase lost its tumor-suppressive effect. Also, S. thermophilus increased the gut abundance of known probiotics, including Bifidobacterium and Lactobacillus via β-galactosidase. β-galactosidase-dependent production of galactose interfered with energy homeostasis to activate oxidative phosphorylation and downregulate the Hippo pathways kinases, which partially mediated the anticancer effects of S. thermophilus.S. thermophilus is a novel prophylactics for CRC prevention in mice. The tumor-suppressive effect of S. thermophilus is mediated at least by the secretion of β-galactosidase.