Objective: This case-control study aimed to assess the impact of Clostridium butyricum triple viable (CBTV) on the performance and gut microbiota of athletes. Methods: We recruited 47 athletes from various sports disciplines and divided them into two groups: Group A received a standard fitness regimen, while Group B received the same fitness regimen along with CBTV supplementation for a period of 4 weeks. Performance measurements were recorded, and gut microbiota analysis was conducted using 16S rRNA sequencing and bioinformatics. Results: After 4 weeks, there were no significant differences in the performance measurements between the two groups (Group A: 159.1±42.4 vs. Group B: 150.8±34.8, p = 0.42). However, the quality of life improvement in Group B was significantly higher than in Group A (Group A: 86.2±26.2 vs. Group B: 89.7±40.7, p < 0.01). Additionally, the gut microbiota analysis revealed that certain bacterial species, including Megamanos, Pseudonocardia, Corynebacterium, and Veillonell, were less abundant in Group B compared to Group A after 4 weeks. Conclusion: This case-control study suggests that CBTV supplementation can enhance the quality of life and influence the abundance of specific bacteria, including a reduction in Megamanos, in athletes. These findings lay the groundwork for further research into the mechanisms by which probiotic bacteria impact gut microbiota in the context of sports performance.