Inflammatory processes are increasingly attributed to macrophage polarization. Proinflammatory macrophages promote T helper (Th) 1 response, tissue repair, and Th2 responses. Detection of macrophages in tissue sections is facilitated by CD68. Our study is focused on the expression of CD68 and the estimation of proinflammatory cytokines in children’s patients with chronic tonsillitis secondary to vitamin D supplementation. This hospital-based Randomized prospective case–control study was conducted on 80 children with chronic tonsillitis associated with vitamin D deficiency where (40 received vitamin D 50,000 IU weekly for 3–6 months and 40 received 5 ml distilled water as placebo). The serum 25-hydroxyvitamin D [25(OH)D] was measured using an Enzyme-linked immunosorbent assay on all included children. Different histological and immunohistochemical studies for the detection of CD68 were done. There was a significantly lower serum level of 25(OH)D in the placebo group versus the vitamin D group (P< 0.001). The levels of pro-inflammatory cytokines, TNFα, and IL-2 significantly increased in the placebo group as compared to the vitamin D group (P< 0.001). The increased level of IL-4 and IL-10 in the placebo group as compared to the vitamin D group was insignificant (P= 0.32, 0.82) respectively. Vitamin D supplementation alleviated the deleterious effect of chronic tonsillitis on the histological structure of the tonsil. Tonsillar tissues of the children in the control and vitamin D groups demonstrated a highly statistically significantly lower number of CD68 immunoexpressing cells compared with those in the placebo group (P< 0.001). Low vitamin D may play a role in chronic tonsillitis. Vitamin D supplementation could help reduce the occurrence of chronic tonsillitis in susceptible children.