Ulcerative colitis (UC) is a chronic recurrent inflammation of the colon. It has been proposed that the UC pathogenesis may be related to vitamin D deficiency and/or vitamin D administration in UC patients may have an ameliorating effect on the intestinal inflammation. The aim of this study was to assess the effect of vitamin D on the serum levels of immune cytokines in UC patients. In this double‐blind randomized controlled trial, 90 mild‐to‐moderate UC patients were assigned to get either a single muscular injection of 7.5 mg vitamin D3 or 1 mL normal saline as placebo. Three months later serum levels of IL‐4, IL‐10, IL‐12p70, IFN‐γ, and TNF‐α were measured. Two group variables were compared using independent t‐test and analysis of covariance (ANCOVA). There was a significant increase in vitamin D only in the vitamin D group. Compared to placebo, vitamin D had significant decreasing effects on serum TNF‐α, IFN‐γ, and IL12p70 levels, but it had no significant effect on serum levels of IL4 and IL10. Vitamin D seems to inhibit Th1 immune responses and have no effect on Th2 responses. The findings of this study support several in vitro studies, which suggest a therapeutic immunomodulatory potential of vitamin D.