(1) Objective: This prospective case-control study aimed to assess the level of serum vitamin D comparing pediatric non-allergic patients with obstructive sleep apnea (OSA) and healthy controls. (2) Methods: The period of the enrollment was from November 2021 to February 2022. Children with uncomplicated OSA caused by adenotonsillar hypertrophy (ATH) were recruited. Allergy was excluded by skin prick test (SPT), and the determination of serum IgE level using ELISA test. Plasma concentration of 25-hydroxy vitamin D (25-OHD) was quantitatively determined; then, the vitamin D concentration in patients was compared with healthy controls matched for sex, age, ethnicity, and characteristics. (3) Results: Plasma 25-OHD levels were significantly lower in patients than in healthy subjects (mean 17 ng/mL, 6.27 DS, range 6–30.7 ng/mL, vs. mean 22 ng/mL, 9.45 DS, range 7–41.2 ng/ ml; p p p-value not significant), might suggest a negative effect of vitamin D deficit on the immune system.