Increasing studies have shown that the efficacy of Weizmannia coagulans in treating various cancers. We recently identified W. coagulans MZY531 with potent cell anti-proliferation and exhibiting apoptosis induction activities against the mouse H22 hepatocellular carcinoma cell line.However, the anti-cancer effect of W. coagulans MZY531 against liver cancer in vivo has not been verified. The objective of this study was to assess the anti-hepatoma effect of W. coagulans MZY531 on H22 tumor-bearing mice and the underlying mechanism. The results demonstrated that W. coagulans MZY531 reduced the weight and size of the tumor in comparison to the model group. The levels of serum pro-inflammatory cytokines, including IL-1β, IL-6, IL-2 and TNF-α were suppressed by W. coagulans MZY531 administration. Immunofluorescence and TUNEL analyses demonstrated that W. coagulans MZY531 significantly increased the number of cleaved caspase-3 cells and induced apoptosis in tumor tissues. Importantly, W. coagulans MZY531 activated the AMPK/mTOR autophagy-dependent apoptosis pathway, and regulated the TLR4/MyD88/TRAF-6/NF-κB and JAK2/STAT3 inflammatory signaling pathways through mechanisms. Additionally, Fecal analysis demonstrated the capacity of W. coagulans MZY531 to remodel the gut microbiota of hepatocellular carcinoma-infected mice. Collectively, this experimental finding suggested that W. coagulans MZY531 exhibited prominent anticancer activities in vivo at least partly via reducing inflammation, inducing autophagy-dependent apoptosis, and regulating gut microbiota in H22 tumor-bearing mice.