Background: With established efficacy and safety, BNT162b2 was one of the first licensed mRNA vaccines against SARS-CoV-2. However, it is unclear whether vitamin D serum concentrations affect vaccine efficacy after three doses. Methods: This prospective, single-center study included 273 staff members (85.4% females) of the National Cancer Comprehensive Center in Slovakia. Median age of participants was 47 years (range: 22–80) and median body mass index (BMI) was 24.8 (16.3–44.6) kg/m². The primary objective was to determine the efficacy of BNT162b2 defined as no Covid-19 disease ≥7 days after its administration. The secondary objective was to monitor IgG neutralizing antibodies and total vitamin D in serum after vaccination and test the hypothesis on correlation of total vitamin D concentrations with the vaccine efficacy. If appropriate, results were stratified by gender (males vs females) and BMI ( < 30 vs≥30 kg/m²). Results: At median follow-up of 4.7 (2.9–5.0) months, vaccine efficacy was 86.1%. The most common adverse events (AEs) were injection site pain (73.3%), fatigue (44.0%), and limb pain (33.0%). No grade 3/4 AEs were observed. Median time from vaccine administration to measurement of IgG and vitamin D concentrations was 3.4 (2.1-4.8) months. IgG concentrations were significantly higher in males compared to females, as well as in subjects with high vs low BMI (p< 0.0004 and p< 0.0001, respectively). Total vitamin D concentrations were significantly lower in individuals with high vs low BMI (p< 0.0144). No differences in the rate of Covid-19 positivity were identified based on gender, BMI, and total vitamin D concentrations. Conclusions: This study confirmed high efficacy of third dose of the BNT162b2 vaccine with an acceptable safety profile. The vaccine efficacy was not affected by gender, BMI, and total vitamin D concentrations. These results suggest that serum vitamin D monitoring after third BNT162b2 dose is not beneficial for an estimation of its efficacy. This study was supported by the National Cancer Institute, Bratislava, Slovakia & OncoReSearch, Rovinka, Slovakia. Keywords: BNT162b2; total vitamin D; efficacy; safety profile; IgG.