To observe the colonic tumor status in mice with azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colitis-associated colorectal cancer (CAC) following autologous fecal microbiota transplantation (AFMT) intervention, and to investigate the anti-tumor effects and underlying mechanism of AFMT through its influence on tight junction proteins. Methods: Female BALB/c mice (SPF grade) were randomly divided into three groups (n=8 each): the Blank Control group, the CAC Model group, and the AFMT Intervention group. The CAC Model group received a single intraperitoneal injection of AOM (10 mg/kg), followed by drinking water containing 3.5% DSS for 7 days, and then 0% DSS for 14 days, constituting one cycle; three cycles were performed to establish the CAC model. The AFMT Intervention group received daily AFMT (0.1 mL autologous fecal suspension) via gavage every other day concurrent with the CAC modeling protocol until cycle completion. The Blank Control group received no special treatment. The general condition and body weight of the mice were monitored. Upon completion of the experiment, colon length was measured and tumor numbers were recorded. Colon pathological examinations were performed. The mRNA and protein expression levels of tight junction proteins Occludin (Ocln) and Claudin-1 (Cldn1) in colorectal tumor tissue were detected using RT-qPCR and Western blotting, respectively. Fecal microbiota composition from each group was analyzed using 16S rRNA gene sequencing. Results: Compared to the Blank Control group, the CAC Model group exhibited significantly shortened colon length and a significant increase in tumor number. After AFMT intervention, the shortening of colon length was alleviated and the tumor number decreased significantly compared to the CAC Model group. Histological examination revealed distorted crypt architecture, reduced goblet cells, and increased inflammatory cell infiltration in the colons of the CAC Model group. Following AFMT intervention, crypt architecture and goblet cell numbers showed improvement, with decreased inflammatory cells. Compared to the Blank Control group, the mRNA and protein expression levels of Ocln (Occludin) and Cldn1 (Claudin-1) were significantly reduced in colorectal tumor tissue of the CAC Model group. AFMT intervention significantly increased the mRNA and protein expression levels of Ocln (Occludin) and Cldn1 (Claudin-1) compared to the CAC Model group. Fecal microbiota analysis showed no statistically significant difference in Firmicutes abundance between the AFMT Intervention group and the Blank Control group, while the CAC Model group had a significantly lower Firmicutes abundance compared to the Blank Control group. No statistically significant difference in Bacteroroidota abundance was found between the AFMT Intervention group and the Blank Control group, but the CAC Model group exhibited significantly higher Bacteroroidota abundance than the Blank Control group. Conclusion: AFMT intervention can restore Firmicutes abundance and reduce Bacteroroidota abundance in the gut, effectively improve the aberrant expression of tight junction proteins within the tumor microenvironment, repair the intestinal barrier, regulate the intestinal functional barrier, alleviate intestinal inflammation, and consequently mitigate the pathological progression of CAC.