Changes in the gut microbiome can be induced by eating habits or alterations in ecological factors, and is proved engaged in colorectal cancer. Thioproline is an antioxidant, and used to be a food additive against aging. This study tries to investigate the impacts of thioproline in restoring the gut microbiota aginst the colorectal cancer. α-diversity and β-diversity analyses of gut microbiota indicate that thioproline can alter the gut microbiota structure of colorectal cancer mice, bringing it closer to that of healthy mice. Analysis of changes at the phylum and genus levels suggests that thioproline can modulate the proportions of Firmicutes and Bacteroidetes, while also increasing the abundance of Muribaculaceae, Lactobacillaceae, and Bacteroidaceae at family level, Muribaculum, [Eubacterium]_xylanophilum_group, and Roseburia at genus level. Functional analysis reveals that oral administration of thioproline influences cancer-related metabolic pathways by altering the composition of gut microbiota in cancerous mice. This study reveals that thioproline exerts anti-colorectal cancer effects by altering the gut microbiota structure in cancerous mice.