Exercise-induced fatigue (EF) refers to the physiological processes that impair the body’s ability to maintain desired levels of functioning and sustain predetermined exercise intensity. It encompasses peripheral discomfort (e.g., muscle soreness, weakness) and central exhaustion, characterized by emotional and sleep disturbances. The detrimental effects of EF significantly impact human productivity and lifestyle. Given the crosstalk between the gut microbiota and physiological metabolism in exercise, there is a growing interest in investigating the role of gut microbiota in elucidating the mechanisms underlying EF. Furthermore, exploring dietary interventions as a novel approach to alleviate EF has gained attention. Our work comprehensively synthesizes the underlying mechanisms and the alterations of gut microbiota community structure and function caused by EF, and evaluates existing evidence on the potential function of probiotics in the regulation of EF. This review aims to enhance our understanding of the correlation between gut microbiome and EF and explores the potential use of probiotics in mitigating EF.