BACKGROUND: Emerging evidence has shown that vitamin D and magnesium have anti-inflammatory and anti-depressant effects. Dietary intake of magnesium is associated with reduced body mass index, waist circumference, body fat percentage, as well as inflammatory biomarkers and depressive symptoms. Vitamin D deficiency has been linked to inflammation, obesity, and depressive symptoms. This study will test the effects of vitamin D and magnesium co-supplementation on mood, serum level of brain-derived neurotrophic factor (BDNF), inflammation, and sirtuin 1 (SIRT1) in obese women. METHODS: We will conduct an 8-week, double-blind, randomized, placebo-controlled clinical trial, in a factorial design, to evaluate the individual effects of vitamin D and magnesium, and co-supplementation of them, on mood, serum level of BDNF, inflammation, and SIRT1 in 108 obese women. DISCUSSION: We hypothesize that vitamin D and magnesium co-supplementation may provide a new adjuvant therapy through modulation of BDNF, inflammation, and SIRT1 in obese women. TRIAL REGISTRATION: Iranian Registry of Clinical Trials, IRCT20090822002365N23. Registered on 16 August 2019.