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Background: Colorectal cancer (CRC) is the third most common malignant tumor worldwide. Emerging evidence suggests that dysbiosis of the colon microbiome may be involved in CRC development. Objectives: The present study aimed to compare the composition and diversity of the colon microbiome by high-throughput 16S ribosomal RNA (rRNA) sequencing between CRC patients and healthy controls. Microbiome composition and diversity were also examined based on gender. Methods: The colon microbiome richness and diversity of samples from 17 CRC patients and 13 healthy controls were analyzed by 16S rRNA sequencing. Alpha and beta diversity were calculated to determine the differences in colon microbiome diversity. Results: Alpha and beta diversity showed significant differences between the CRC and healthy control groups regarding the microbiome. Our results showed that CRC samples had the highest richness and diversity. The total number (P ≤ 0.01), phylogenetic diversity (P ≤ 0.01), Chao1 (P ≤ 0.01), Shannon (P ≤ 0.05), and Simpson (P ≤ 0.01) indices were significantly higher in the CRC group than in the healthy control group. In addition, the comparison between females and males showed that the microbiome diversity was higher in the CRC female (CRC-F) group than in other groups. Prevotella, Fusobacterium, Akkermansia, Leptotrichia, Streptococcus, and Parabacteroides were more commonly observed in the CRC group, while Bacteroides, Enterobacteriaceae (unknown genus), Ruminococcus, and Campylobacter were more commonly observed in the healthy control group. Conclusions: This study showed differences between the CRC and healthy control groups regarding the diversity and composition of the colon microbiome, suggesting a contribution of the microbiome in the development and progression of CRC.