Abstract: Objective: This study aimed to investigate the therapeutic mechanism of Oviductus Ranae (OR) in chronic fatigue syndrome (CFS). Methods: Active ingredients and potential targets of OR were screened using TCMSP, BATMAN, and GeneCards databases with the keywords "OR" and "CFS." Molecular docking was performed to assess binding affinities between bioactive compounds and their targets. A mouse model of CFS was constructed using an exhausting swim and chronic restraint stress. Mice were treated with OR suspension (800, 400, or 200 mg/kg) via oral gavage for 14 days. Biochemical parameters and inflammatory markers were analyzed in serum, muscle, liver and brain tissue. Results: Five bioactive compounds and eight core targets associated with CFS improvement were identified. OR exerts its therapeutic effect primarily through modulation of the IL-17/TRAF6 signaling pathway. Molecular docking indicates that campesterol, a component of OR, exhibits the strongest binding affinity, suggesting its role as a key active ingredient. Results from in vivo animal experiments demonstrated that OR had a significant therapeutic effect on CFS model mice, effectively improving their behavioural indicators and resolving their physiological and biochemical abnormalities. In terms of behavioural aspects, the mice in the administered group showed significant increases in body weight and diet, as well as a significant increase in sugar-water preference (P<0.01 or P<0.05), compared with the model group. Several behavioural tests revealed that, in the forced swimming test (FST) and the tail suspension test (TST), the administered group's immobility time was significantly shorter (P<0.01). In the open-field test (OFT), locomotion speed, total locomotion distance and the number of times standing up were all significantly increased (P<0.01 or P<0.05). In the Morris Water Maze (MWM), avoidance latency was significantly shorter. Concurrently, the movement distance and residence time in the target quadrant were significantly prolonged (P<0.01 or P<0.05). The assay results of the biochemical indicator showed that BUN levels, IL-6 levels, TNF-α levels, COX2 levels and MDA levels were significantly reduced, while SOD activity and GSH-Px activity were significantly increased in the administered group of mice (P<0.01 or P<0.05). Additionally, lactic acid content in muscle tissue decreased significantly (P<0.01 or P<0.05). Studies of the molecular mechanism suggest that OR may exert therapeutic effects by modulating the IL-17/TRAF6 signalling pathway. Conclusions: OR ameliorates CFS by modulating the IL-17/TRAF6 signaling pathway, thereby providing a novel perspective for the clinical management of CFS.