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Background and Aim: Vitamin D (VD) deficiency was reported to correlate with ulcerative colitis (UC) activity, which might be closely related to gut microbiota dysbiosis. This study aims to investigate the effects of washed microbiota transplantation (WMT) on VD metabolism in UC., Methods: The serum levels of 25-hdroxyvitamin D [25(OH)D] in 121 patients with UC and 53 healthy controls (HC) were detected. Subsequently, a non-randomized control trial (non-RCT) was conducted. Patients with UC were non-randomly assigned to undergo WMT (n = 28) vs. conventional treatment (5-aminosalicylic acid, 5-ASA, n = 10). Serum levels of 25(OH)D, fecal microbiota, and the expression of vitamin D receptor (VDR) in patients with UC were evaluated with a 3-month follow-up., Results: Serum VD levels collected in the clinic practice indicated that patients with UC had significantly lower VD levels than HC (P < 0.001). In the non-RCT, serum 25(OH)D level and VDR expression significantly increased (P = 0.011, 0.026, respectively) in the WMT group, while no noticeable changes were observed in the non-WMT group. Microbiome profiling revealed that the increase in VD levels after WMT was positively associated with the abundances of Adlercreutzia&#95;equolifaciens, Ruminococcus&#95;obeum, and Dorea but negatively correlated with Escherichia., Conclusions: The study suggested that WMT increases the levels of VD with characteristic changes of specific microbiota, which indicated the association between the VD and the activity of UC might be regulated by gut microbiota., (© 2024 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)