The only reliable factor that reduces the risk of colorectal carcinogenesis is physical activity. However, the underlying mechanisms remain unclear. In this study, we examined the effects of physical activity against gut microbiota, including mucosa-associated microbiota (MAM) on azoxymethane-induced colorectal tumors in obese mice. We divided the subjects into four groups: normal diet (ND), high-fat diet (HFD), ND + exercise (Ex), and HFD + Ex groups. The Ex group performed treadmill exercise for 20 weeks. Thereafter, fecal and colonic mucus samples were extracted for microbiota analysis. DNA was collected from feces and colonic mucosa, and V3–V4 amplicon sequencing analysis of the 16SrRNA gene was performed using MiSeq. The HFD group had significantly more colonic polyps than the ND group (ND 6.5 ± 1.3, HFD 11.4 ± 1.5, p < 0.001), and the addition of Ex suppressed the number of colonic polyps in ND and HFD groups (ND 6.5 ± 1.3, ND + Ex 2.8 ± 2.5, p < 0.05). The HFD group showed significantly lower concentrations of succinic, acetic, butyric, and propionic acids (mg/g) in feces, compared with the ND group (succinic acid HFD 0.59, ND 0.17; acetic acid HFD 0.63, ND 2.41; propionic acid HFD 0.10, ND 0.47; and N-butyric acid HFD 0.31, ND 0.93). In the case of ND, succinic acid and butyric acid tended to decrease with Ex (succinic acid ND 0.17, ND + Ex 0.12; N-butyric acid ND 0.93, ND + Ex 0.74 0.74). Succinic acid, acetic acid, butyric acid, and propionic acid levels in feces were significantly lower in the HFD group than in the ND group; in both feces and mucus samples, Butyricicoccus and Lactobacillus levels were significantly lower in the HFD group. Akkermansia was significantly increased in ND + Ex and HFD + Ex groups. Diet and exercise affected the number of colorectal tumors. Furthermore, diet and exercise alter intestinal MAM, which may be involved in colorectal tumor development.