Abstract. The present randomized, double-blind, placebo controlled study aimed to evaluate the effect of vitamin D supplementation on matrix metalloproteinases-2, -9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in subjects with metabolic syndrome. Forty-six eligible subjects were randomly assigned to either vitamin D or placebo groups for 16 weeks. The participants were asked to take 50,000 IU vitamin D or matching placebo every week. Metabolic and anthropometric indices, serum MMP-2, MMP-9, TIMP-1 and high-sensitivity C-reactive protein (hsCRP) were assessed before and after intervention. Moreover, dietary intake, sun exposure and physical activity were also determined. The trial was registered at http://www.irct.ir (No. IRCT201409033140N14). Participants were 40.20 ± 4.60 y and 45.50% males. Compared to the baseline values, MMP-9 and TIMP-1 concentrations were decreased after 16 weeks in the intervention group (p = 0.03 and p = 0.04, respectively). However, the changes of MMP-2, MMP-9, TIMP-1 and hsCRP in the intervention group were not significant compared to the placebo group (p > 0.05). Furthermore, the metabolic or anthropometric indices between two study groups remained unchanged (p > 0.05). The findings of the present study demonstrated no effect of vitamin D supplementation on MMP-2, MMP-9 and TIMP-1 concentrations in subjects with metabolic syndrome. However, there is a need for more longitudinal trials to investigate the role of vitamin D on atherosclerosis and cardiovascular diseases in subjects with metabolic syndrome.