Cancer-related fatigue (CRF) is one of the most common and serious complications in cancer patients, which greatly reduces the quality of life. The mechanism induced fatigue may be diverse. In this study, we tried to investigate the effect of 1,25(OH)₂D₃, the active metabolite of vitamin D on CRF in Lewis lung cancer-bearing mice. Network pharmacological analysis, behavioral testing, western blotting, ELISA and flow cytometry were used. We found that there was an interaction between proteins related to the role of 1,25(OH)₂D₃ and CRF-related proteins. The results of animal model experiments showed that 1,25(OH)₂D₃ could mitigate the CRF behavior of tumor-bearing mice, and the treatment of 1,25(OH)₂D₃ reduced the levels of inflammatory factors, changed the tryptophan metabolism direction, and caused changes in immune cells. Collectively, 1,25(OH)₂D₃ might improve CRF in tumor-bearing mice by changing the direction of tryptophan metabolism and inflammatory factor levels. This study provided a possible solution for patients with clinical CRF.