Ammonia (NH3) is an irritant and harmful gas. Its accumulation in the poultry house poses detrimental effects on the respiratory mucosal system of birds. In this process, the relative humidity of the poultry house also plays an important role in potentiating the adverse effects of NH3 on the respiratory status of birds, causing severe physiological consequences. In this study, the combined effects of NH3 and humidity on the respiratory mucosal barrier of laying hens was studied. The gene expression of tight junction proteins, mucin, inflammatory cytokines secreted by Th1/Th2 cells, and proteins related to the Nuclear factor-κB (NF-κB) signaling pathway were detected by qRT-PCR. In addition, the contents of mucin and secretory immunoglobulin A (SIgA) in bronchoalveolar lavage fluid (BALF) were determined. The results showed that treatment with NH3 alone or NH3 and humidity led to morphological changes in the respiratory tract, decreased the gene expressions of tight junction protein, and increased the expression of mucin. Also, the expression of interleukin-4 (IL-4) and IL-10 were increased, whereas, the expression of interferon-γ (IFN-γ) and IL-2 was decreased in laying hens treated with NH3 and humidity. Furthermore, the activation of inhibitor kappa B kinase β (I-KK-β) and the degradation of inhibitor of NF-κB α (I-κB-α) contributed to the activation of the NF-κB pathway, such that the downstream genes, cycooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS) were significantly increased. In conclusion, NH3 damaged the mucosal barrier and induced an imbalance in the mucosal immunity, leading to respiratory tract inflammation. Thus, the relative humidity of the environment aggravates the adverse effects of NH3 in poultry.