Scope Salidroside (SA) is an active compound derived from Rhodiola rosea and is widely used in healthcare foods. However, the underlying mechanism and its specific role in regulating the gut microbial community during exercise (Ex) remains unknown. Methods and results Mice are subjected to a weight‐loaded swimming test (WST) Ex to determine how gut microbiota affects the antifatigue activity of SA. The SA‐treated group mice (100 mg kg−1.bw.) display a significant increase in swimming time compared to the control group (26.2 versus 10.5 min, p < 0.01), as well as an increase in respiratory enzymatic activities after swimming. The respiratory enzymatic activities are significantly higher in the SA‐treated group than in the RS (regular rest) group after swimming. The bacteria profiles in the Ex + SA group change significantly with higher species diversity and abundance. Receiver operating characteristic (ROC) curves of Alistipes, Rikenellaceae, Parabacteroides, Candidatus Arthromitus, and Lactobacillus indicate a high diagnostic utility to distinguish SA treatment. Microbial function analysis shows that SA may improve Ex‐induced fatigue by modulating energy metabolism‐related processes. Conclusions SA demonstrates antifatigue effects on various levels of regulating energy metabolism and microbial composition, providing insights into the underlying mechanisms of SA as a natural prebiotic. Salidroside (SA) is an active compound derived from Rhodiola rosea, which could enhance the motor performance and alleviate host's exercise (Ex)‐induced injuries. The antifatigue effect of SA is probably associated with its specific role in regulating gut microbial composition and functions via bacterial metabolic pathways during Ex.