Airway epithelium is an active and important component of the immunological response in the pathophysiology of obstructive lung diseases. Recent studies suggest an important role for vitamin D3 in asthma severity and treatment response. Our study evaluated the influence of an active form of vitamin D3 on the expression of selected mediators of allergic inflammation in the respiratory epithelium. Primary nasal and bronchial epithelial cells were exposed to1,25D3 for 1 hour and were then stimulated or not with IL-4, TNF-α, LPS, and poly I:C. After 24 hours TSLP, IL-33, and IL-25 protein levels were measured in culture supernatants using ELISA and mRNA levels in cells by real time PCR. 1,25D3 increased TSLP concentration in unstimulated nasal epithelial cells, but did not influence IL-33 and IL-25 expression. In IL-4-stimulated epithelial cell cultures 1,25D3 mostly inhibited TSLP and IL-33 expression. In LPS-treated cultures 1,25D3 decreased IL-33 expression. Simultaneously 1,25D3 augmented IL-25 production in the same model of stimulation. Our study revealed the dual nature of vitamin D3 manifested in both pro- and anti-inflammatory properties observed in airway epithelial cells.