Background: Vitamin D has been suggested to influence the immune system, and vitamin D metabolites and the vitamin D receptor (VDR) are generated and expressed in white blood cells (WBC). Moreover, vitamin D status has been associated with incidence and prognosis of some respiratory tract infections (RTI). Therefore, we investigated the effect of vitamin D₃ supplementation on WBC, acute phase reactants (APR), and the risk of developing RTIs. Methods: A double-blinded, randomized, placebo-controlled clinical trial of 307 infertile men with multiple secondary immunological endpoints. The vitamin D₃ group (n = 151) initially received 300,000 IU (7,500 µg) cholecalciferol once - followed by 1,400 IU (35 µg) daily for 150 days. The placebo group (n = 156) did not receive active ingredients. Results: At baseline, stratification into clinically relevant groups of vitamin D status (< 25; 25–50; 50–75; >75 nmol/L), showed an inverse association with total leucocyte concentrations (7.0 vs. 6.0 vs. 6.0 vs. 5.5 (10⁹/L); p = 0.007), lymphocytes (2.4 vs. 2.1 vs. 2.0 vs. 2.0 (10⁹/L); p = 0.048), CRP (2.0 vs. 1.7 vs. 1.2 vs. 1.2 (mg/L); p = 0.037), and orosomucoid (0.82 vs. 0.77 vs. 0.76 vs. 0.70 (g/L); p = 0.015). After 150 days, no differences were detected in WBC counts or APRs between the vitamin D₃ and the placebo group. However, vitamin D₃ treated men had a higher prevalence of self-reported RTIs compared with the placebo group (55% vs. 39%; p = 0.005). Conclusions: High-dose vitamin D₃ supplementation did not alter WBCs or APRs, but a higher prevalence of respiratory infections was observed in the vitamin D₃ group. Serum 25(OH)D₃ was negatively correlated with most WBCs, indicating that vitamin D status may be linked with inflammation and WBC turnover, but not an important determinant of developing RTIs. Trial registration: NCT01304927 (ClinicalTrials.gov). Registered February 20, 2011.