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Purpose: This randomized clinical trial (RCT) was designed to assess the effect of VD3, n-3FA, and their combination on serum leptin levels in people with vitamin D deficiency (VDD). Subjects and methods: One hundred and forty six participants, were randomly assigned into four groups supplemented with the dose of 50,000 IU VD3 taken weekly (D), 300 mg n-3FA taken daily (Om), and their combination (D+Om) or control (C) for eight weeks. Fasting baseline and follow-up (10 weeks; 8 weeks supplementation plus washout period of 2 weeks) of serum 25 hydroxyvitamin D (25OHD), leptin, glucose, triglycerides (TG), parathyroid hormone (PTH), calcium, and phosphorus were assayed. A paired T-test was used to assess the changes in serum leptin levels over of the follow-up period. Results: Significant increase in follow-up serum leptin (10.62 ± 7.18 to 14.42 ± 8.29 ng/mL, P = 0.002) and TG (154 ± 84.4 to 200.1 ± 79, P = 0.015) levels were observed in n-3-FA supplemented group. Combination therapy (VD3 plus n-3 FA) significantly increased serum 25OHD (13.49 ± 4.64 to 37.09 ± 11.13 ng/mL, P < 0.001), TG levels (114.3 ± 57.3 to 139.1 ± 60.7 mg/mL, P = 0.007) and insignificantly serum leptin (6.74 ± 4.87 to 8.01 ± 6.77 ng/mL, P = 0.269). Conclusion: Our study referred that notable elevation in leptin and TG levels might be linked to leptin resistance. However, further RCTs are required to clarify possible consequences resulted from the extensive administration of n-3FA supplements and their combinations with high doses of VD3 supplements on humans’ health.