Ammonia (NH), an inhaled harmful gas, is not only an important volatile in fertilizer production and ranching, but also the main basic component of haze. However, the effect and mechanism of NH on the intestines are still unclear. To investigate the intestinal toxicity of NH inhalation, morphological changes, transcriptome profiles and oxidative stress indicators of jejunum in broiler chicken exposed to NH for 42 days were examined. Results of morphological observation showed that NH exposure caused deficiency of jejunal microvilli and neutrophil infiltration. Transcriptomics sequencing identified 677 differential expressed genes (DEGs) including 358 up-regulated genes and 319 down-regulated genes. Enrichment analysis of obtained DEGs by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) found that biological functions and pathways affected by NH included antioxidant function, inflammation, microtubule and nutrition transport. Relative genes validation and chemical detection confirmed that NH-induced oxidative stress by activating CYPs and inhibiting antioxidant enzymes promoted inflammatory response and decreased microtubule activity, thus destroying the balance of nutritional transporters. Our study perfects the injurious mechanism of NH exposure and provides a new insight and method for environmental risk assessment.