ObjectiveTo investigate the effects of oral probiotics on gut microbiota diversity, colony structure, and intergroup differences in mice with subcutaneous colon cancer tumors, based on 16S rRNA sequencing technology. Methods Twenty-four 6-week-old male BALB/ c mice were divided randomly into normal control group (NC group, n= 8), model group (M group, n= 8), and probiotic + model group (PM group, n= 8) after adaptive feeding for 1 week. Mice in the PM group were given 200 μL probiotic mixed solution (Bifidobacterium longum and Lactobacillus delbrueckii subsp. bulgaricus mixed lyophilized powder, 2 × 10⁸ colony-forming units) by gavage three times/ week for 7 weeks, while the M group and PM group received 200 μL normal saline. At 10 weeks old, 0. 2 mL CT26.WT cell suspension (1 × 10⁷ / mL) was inoculated subcutaneously into the left hind limbs of M group and PM group, while NC group were inoculated with 0. 2 mL normal saline. The general state of mice was observed, the growth of subcutaneous tumor was monitored, and the changes of intestinal flora structure were detected by 16S rRNA sequencing. Results The subcutaneous tumors of the M group were prominent, and the subcutaneous tumor volume and weight of the PM group were significantly reduced (P<0. 05). Compared with NC group, Alpha diversity index was lower in the M group, and a significant difference of Beta diversity inter groups (P< 0. 01). And supplementation of probiotics had a certain effect on gut microbiota diversity in the M group. Compared with M group, the relative abundance of Bacteroidetes, Proteobacteria, Muribaculaceae,Bacteroides were higher in the PM group, while the relative abundance of Firmicutes, Desulfobacterota, Lachnospiraceae_NK4A136_ group, Alistipes were lower in the PM group. LEfSe analysis showed that Muribaculaceae and Bacteroides in the PM group were different species with high abundance (LDA values > 4). Conclusions Oral probiotics may improve the gut microbiota by increasing the relative abundance of beneficial Muribaculaceae and Bacteroides in subcutaneous tumors in mice with colon cancer.