Ammonia (NH3) is an environmental contaminant that is causing increasing problems with human and animal health due to the development of poultry industry. There are limited studies on the effect of NH3 inhalation toxicity on the intestinal tract of animals, and underlying molecular mechanisms remain unclear. In the present study, we established a chicken model of NH3 aspiration-induced injury for 42 days and observed histopathological changes of the jejunum. Tandem mass tag-based quantitative proteomic analysis was applied to investigate changes in the protein profile in the jejunum tissue of chickens that were exposed to NH3. Overall, 48 significantly differentially expressed proteins (DEPs) were identified. GO and KEGG analyses revealed that most DEPs were closely related to epithelial-to-mesenchymal transition (EMT), cell-cell junctions, and fibrosis-related factors. Regarding fibrosis, type I collagen and fibronectin were significantly increased. With respect to EMT, epithelial marker proteins (such as E-cadherin and keratin) were repressed, while mesenchymal marker proteins (such as vimentin) were activated. Loss of epithelial cell-cell junctions (such as tight junctions, adherens junctions and desmosomes) were observed. Additionally, overexpression of transforming growth factor-beta (TGF-β) may play a key role in the EMT process and fibrosis. Taken together, these findings suggested that NH3 triggered the EMT and disassembly of epithelial cell-cell contacts, resulting in jejunal fibrosis that was mediated by TGF-β in chickens. The results of our study will contribute to provide a technical reference regarding the research methods of intestinal toxicity of NH3 and have largely regulatory implications for ecological risk assessment of human health.