Background:Vitiligo is an autoimmune pigmentary disorder in which vitamin D may influence melanocyte survival, melanogenesis, and immune regulation. However, studies evaluating the association between vitamin D deficiency and vitiligo have reported inconsistent findings.Objectives: To compare serum 25-hydroxyvitamin D [25(OH)D] levels between patients with non-segmental vitiligo and healthy controls and to evaluate their association with clinical characteristics of vitiligo.Materials and Methods: A prospective case–control study was conducted at a tertiary care hospital in North India from February 2025 to February 2026. Thirty treatment-naïve patients with non-segmental vitiligo and 30 age- and sex-matched healthy controls were enrolled. Serum 25(OH)D levels were measured using electrochemiluminescence. Vitamin D status was categorized as deficient (<20 ng/mL), insufficient (20–30 ng/mL), and sufficient (31–70 ng/mL). Statistical analysis was performed using SPSS, with P <0.05 considered statistically significant.Results: The mean age of patients with vitiligo was 26.67 ± 16.20 years. Mean serum 25(OH)D levels were significantly lower in cases than in controls (19.6 ± 10.9 ng/mL vs. 27.6 ± 12.7 ng/mL; P = 0.021). Vitamin D deficiency was observed in 56.7% of patients with vitiligo compared with 23.3% of controls, while sufficient vitamin D levels were present in 23.3% and 53.3%, respectively. No significant association was observed between serum vitamin D levels and sex, clinical type of vitiligo, or associated autoimmune diseases.Conclusion:Patients with non-segmental vitiligo had significantly lower serum 25(OH)D levels than healthy controls, supporting an association between vitamin D deficiency and vitiligo. Assessment of vitamin D status at diagnosis may be beneficial, and supplementation should be considered in deficient individuals. Larger longitudinal studies are required to clarify whether correction of vitamin D deficiency influences disease onset, progression, or treatment outcomes.