Introduction: Vitamin D3 plays an important role in the immune system, and its deficiency has been implicated in various skin diseases, including atopic dermatitis and psoriasis. Acne is a common inflammatory skin disease of adolescence and young adulthood that affects the face and upper trunk. The scarring caused by acne can have a significant psychological impact on patients and greatly affect their quality of life. However, the association between the severity of acne and vitamin D3 levels remains unclear. Aim: To assess the role of vitamin D3 in the severity of acne vulgaris in adolescents. Materials and Methods: This prospective cross-sectional study was conducted at the Department of Dermatology and Biochemistry, VIMSAR, Burla, Sambalpur, Odisha, India, from August 2022 to November 2022. Adolescents between the ages of 10 and 19 years, of either sex, with acne were clinically examined, and the severity of acne was evaluated using the Global Acne Grading System (GAGS) score. Serum vitamin D3 levels were measured using a Chemiluminescence Immunoassay (CLIA) analyser (Electra FA). Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software version 21.0 (SPSS IBM Corporation, Armonk, New York), and a p-value <0.05 was considered statistically significant. Results: A total of 150 clinically diagnosed cases of acne vulgaris patients were evaluated. The study included 73 males and 77 females. The mean age of males was 19.1±2.3 years, and for females, it was 18.2±2.4 years. Serum vitamin D3 levels were lower in patients with severe acne (18.03 ng/mL) compared to those with moderate acne (23.57 ng/mL) and mild acne (28.02 ng/mL). The association between serum vitamin D3 concentration and the different degrees of acne vulgaris was statistically significant (p<0.05). There was a strong negative correlation between severe acne and serum vitamin D3 levels (r=-0.042, p-value=0.002). Conclusion: Vitamin D3 deficiency was more prevalent in patients with severe acne, and serum vitamin D3 levels were inversely correlated with the severity of acne vulgaris. [ABSTRACT FROM AUTHOR]