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Background Both augmented inflammatory reaction and low vitamin D status are associated with depression but the magnitude of their relationships is unclear. This study was, therefore, conducted to evaluate the effects of vitamin D supplementation on serum 25(OH)D concentration, depression severity and some pro-inflammatory biomarkers in patients with mild to moderate depression. Methods An 8-week double-blind randomized clinical trial (RCT) was performed on 56 (18–60 yrs) patients with mild to moderate depression, randomly assigned to intervention (50,000 IU cholecalciferol 2wks −1 ) and control (placebo) groups. Serum 25(OH)D, intact parathyroid hormone (iPTH), interlukin (IL)-1β, IL-6, high-sensitivity C-reactive protein (hs-CRP) and depression severity (Beck Depression Inventory-II) (BDI-II)) were initially and finally assessed. Results At the end point, statistically significant changes were observed only in intervention group as compared with controls including increased 25(OH)D concentration (+ 40.83 ± 28.57 vs. + 5.14 ± 23.44 nmol L −1 , P  < 0.001) and decreased depression severity (-11.75 ± 6.40 vs. -3.61 ± 10.40, P  = 0.003). No significant within- or between group differences were observed in serum IL-1β, IL-6 and hs-CRP concentrations. Conclusion Increased circulating 25(OH)D concentrations following 8-week vitamin D supplementation (50,000 IU 2wks −1 ) resulted in a significant decrease in BDI-II scores in patients with mild to moderate depression. However, this effect was independent of the serum concentrations of the studied inflammatory biomarkers. Trial registration The clinical trial registration code was obtained from the Iranian Registry of Clinical Trials (date of registration: 17/09/2018, registration number: IRCT20170926036425N1) and ClinicalTrials.gov (date of registration: 04/12/2018, registration number: NCT03766074)