The post hoc analysis presented here aimed to address the influence of endogenous vitamin D in the immunological mechanism underlying effective mite allergoid immunotherapy (AIT). Previously, we have shown that in allergic children, after 12 months of this immunoactive treatment, functionally potentiated memory regulatory T cells are identified. Indeed, AIT is the only known treatment that is able to reshape the detrimental immune response against the allergen into a non-noxious one. Besides, VD is widely considered an immunoregulatory molecule that is endogenously produced and exogenously provided by foods and supplements that might interact with the AIT mechanism, thus affecting its outcome. Therefore, a post hoc analysis of the clinical and immunological data from three different cohorts of allergic patients was performed. One cohort (N = 70) was on a standard symptom-controlling pharmacological treatment, while the other two (N = 60 and N = 35) were treated with AIT for 12 months. In the first cohort, a lower mean endogenous VD level (<22 ng/mL) was observed along with worse symptoms and a greater use of medications. Remarkably, the comparison between two sub-cohorts of patients with a serum VD level above (N = 32) or below (N = 28) a cut-off value set at the mean value (27 ng/mL) revealed that optimal improvement of all clinical and immune parameters was achieved (as expected from effective AIT), irrespective of the VD level. Notably, the third analysis, carried out on one cohort of AIT patients that were also concomitantly taking VD3 as a food supplement (N = 19), was distinguished by an uppermost overall treatment outcome (the amelioration of symptoms, the lowest medication requirements, and a reduction in the total and allergen-specific IgE levels), as well as an increase in the allergen-specific tolerogenic memory T regulatory cells. These findings suggest that the endogenous VD level affects the allergy severity and allergen immunotherapy effectiveness. In addition, VD3 might be investigated as an add-on supplement to obtain the best out of immunotherapy in VD-deficient/-insufficient allergic patients. The immunogenic, but low-allergenic, mite allergoid used as the bioactive agent might have contributed to minimizing the allergic response and highlighting the immunological effects described here.