Objective To 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. Method Twenty-four 6-week-old male BALB/c mice were divided randomly into normal control (NC, n=8), model (M, n=8), and probiotic+model groups (PM, 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 and PM groups 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 and PM mice, while NC mice were inoculated with 0.2 mL normal saline. The general condition of the mice and growth of subcutaneous tumors, and changes in the gut microbiota structure by 16S rRNA sequencing were monitored. 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.