Microtubule-actin cross-linking factor-1 (ACF7, or MACF1) regulates cytoskeletal focal adhesion dynamics and migration in various tissues. High fat diet (HFD) induces gut microbiota dysbiosis and metabolic disorders, and increases intestinal permeability and inflammatory response. Here we investigated the synergistic effects of intestinal ACF7 conditional knockout (ACF7 cKO) and HFD on metabolism phenotypes, gut microbiota and intestinal barrier function in mice. ACF7 cKO and control (ACF7fl/fl) mice (8-week-old) were fed with either chow diet or HFD, for 16 weeks. The increase of body weight and fat pad weight were impaired in HFD-fed ACF7 cKO mice, which can be attributed to decreased food intake and absorption. The metabolic status of HFD-fed ACF7 cKO mice was dramatically changed when compared to the other groups. In addition, HFD-fed ACF7 cKO mice had increased epithelial cell apoptosis, intestinal permeability and inflammatory response when compared with the other groups. The ACF7 cKO-induced changes in alimentation, intestinal barrier function, and gut microbiota were independent of dietary treatment. Taken together, our studies for the first time proved HFD and ACF7 cKO have synergistic damaging effects on intestinal homeostasis. ACF7 is a crucial protective molecule in HFD-induced intestinal diseases.