Abstract: Objective To elucidate the gut microbiota changes and molecular mechanism of Huaier in enhancing the efficacy of oxaliplatin (OXA) chemotherapy in gastric cancer (GC). Methods The effects of Huaier on the gut microbiota structure and intestinal barrier function in MKN45-bearing mice were analyzed using 16S rRNA sequencing, RT-PCR, and IHC. UPLC-MS, network pharmacology, as well as in vivo experimental approaches were employed to systematically investigate the key bioactive constituents in Huaier and elucidate the underlying mechanisms by which it exerts therapeutic effects against GC. The expression of the PI3K/AKT/SREBP pathway was detected in cancer cells and tissues by Western blotting assay. The safety profile of the drug combination was verified through serum biochemistry and H&E-stained tissue section analysis. Results Huaier inhibits the PI3K/AKT/SREBP pathway by increasing the abundance of Akkermansia muciniphila (A. muciniphila), reducing lipid droplet deposition, and ultimately enhancing the sensitivity to OXA in the treatment of GC. Conclusion This study elucidated the novel application of Huaier as a treatment for GC with improved chemosensitivity and provided new ideas for Huaier to play a systemic therapeutic role from the perspectives of molecular mechanisms and gut microbiota.