Background and Objectives: According to the demographic health survey conducted in 2015, Egypt had 10% documented prevalence of anti-HBc positive patients aged 1-59 and 1% viremic patients amongst the population in the same age group, with a domination of genotype D. Several studies claimed the possible role of vitamin D deficiency in hepatitis B virus (HBV) replication and disease progression. Patients and Methods: Serum vitamin D levels [25(OH) D3] were assessed in 96 HBeAg negative non-cirrhotic chronic HBV patients and 25 healthy subjects classified as following: Group I: 48 chronic HBV patients with persistently normal ALT levels and HBV DNA level < 2000 IU/mL for ≥ 6 months; Group II: 48 chronic HBV patients with CHB with persistently elevated ALT and HBV DNA level ≥ 2000 IU/mL for ≥ 6 months; and Group III: 25 apparently healthy subjects with normal liver enzymes and negative hepatitis viral markers were taken as the control group. Results: Vitamin D was much more deficient in group II than in group I and group III being 11.55 ± 3.97 ng/mL, 15.03 ± 3.45, 27.00 ± 6.76 ng/mL (P < 0.001), respectively, and a strong negative correlation was observed between vitamin D levels and HBV DNA levels (P = 0.043) in groups I and II. Conclusion: The current study showed high HBV DNA replication in patients with vitamin D deficiency suggesting the antimicrobial immunomodulatory role of vitamin D.