Objective: The gut microbiota plays a critical role in regulating human health and athletic performance. Probiotic supplementation has been shown to modulate gut microbiota composition and improve exercise performance. This study aimed to investigate the effect of probiotic yogurt supplementation on gut microbiota and its relationship with exercise-related psychological fatigue in female taekwondo athletes. Methods: Twenty female taekwondo athletes were randomly assigned to either a dietary intervention group (DK) or a control group (CK). The athletes’ exercise-related psychological fatigue was measured using the Athlete Burnout Questionnaire (ABQ) before and after an 8-week intervention. High-throughput sequencing was used to profile the gut microbiota, and functional prediction of the microbial community was performed. The effect of the dietary intervention on the athletes’ exercise-related psychological fatigue clearance rate and its relationship with the gut microbiota were explored. Results: (1) The probiotic supplementation ofBifidobacterium animalisssp. lactis BB-12 for 8 weeks significantly increased the ABQ scores of the DK group compared to the CK group (p< 0.05). (2) The abundances ofBifidobacterium,Bacteroides,Lachnospiraceae, family _Lactobacillaceae, and genus _Lactobacilluswere significantly higher in the DK group than in the CK group after probiotic supplementation, whileEscherichia coliwas significantly lower in the DK group than in the CK group. (3) The ABQa scores were positively correlated withProteus; ABQb scores were positively correlated withStreptococcusandEnterococcus; and ABQc scores were positively correlated withKlebsiella,Bacteroides, andStreptomyces. (4) The DK group had significantly higher levels of L-arginine biosynthesis I (via L-ornithine), fatty acid biosynthesis and oxidation, and L-isoleucine biosynthesis III pathways compared to the CK group. Tyrosine degradation I (via 2,3-dihydroxyphenylpropionate) was significantly lower in the DK group than in the CK group. Conclusions: Probiotic yogurt supplementation ofBifidobacterium animalisssp. lactis can promote the clearance of exercise-related psychological fatigue in female taekwondo athletes by upregulating beneficial gut microbiota, inhibiting harmful gut microbiota, and regulating relevant metabolic pathways.