Background: Serum 25(OH)-Vitamin D3 (VitD3) deficiency during infancy has been associated with asthma. The potential therapeutic role of VitD3 given in the airways and its interference with the allergen and Rhinovirus was the objective of this study. Methods: In two cohorts of children with and without asthma, serum levels of the C-reactive protein (CRP) were correlated to Serum VitD3 and in peripheral blood T cell inhibitor marker Programmed cell death protein 1 (PD1) mRNA was analyzed. In a murine model, VitD3 was given intranasally in vivo and in vitro to lung cells with allergen and Rhinovirus. Results: In the cohorts of pre-school age children without (control) asthma, CRP and VitD3 levels inversely correlated. In preschool asthmatic children that did not receive VitD3 supplementation as infant had more episode of asthma exacerbation associated with high CRP serum level. In peripheral blood cells from control but not asthmatic children with higher serum levels of VitD3 had lower PD1 mRNA levels. In murine model, OVA intranasal challenge induced Innate Lymphoid Cells type 2 (ILC2)-associated markers and Eosinophils in BALF and VitD3 inhibited lung inflammation and ILC2 markers. Furthermore, VitD3 given intranasally, induced CD4+T cells and reduced PD1, T regulatory cells in the lung. Similarly, VitD3 had a suppressive role on CD4+PD1+ T cells involved in T cell exhaustion in the airways in the absence of ST2 after Rhinovirus infection. Conclusion: These data support an inhibitory role of VitD3 on T cell exhaustion after allergen and rhinovirus infection that is relevant for pediatric asthma.