Ammonia (NH) is a major gaseous pollutant in livestock production and has adverse effects on production, health and welfare of animals. The liver is one of the target organs of NH, and excessive NH inhalation can induce liver damage. However, the toxicity assessment of NH on pig liver and its mechanism have not been reported yet. Recently, transcriptome analysis has become a major method to study the toxic mechanism of pollutants in environmental toxicology. Therefore, in the present study, we examined the effects of excessive NH inhalation on the liver of fattening pig through chemical analysis, ELISA, transcriptome analysis and real-time quantitative PCR (qRT-PCR). Our results showed that the transcriptome analysis database of fattening pig liver under excessive NH exposure, and 449 differentially expressed genes (DEGs) (including 181 up-regulated DEGs and 168 down-regulated DEGs) were found. Some genes associated with the 3 Gene Ontology (GO) terms (liver function, immune, antioxidant defense) were validated by quantitative real-time PCR. In addition, the activities of GPT and GOT in NH group were significantly increased by 63.5% and 37.4% (P < 0.05), respectively. Our results indicated that NH exposure could cause changes in transcriptional profiles and liver function, and induce liver damage in fattening pigs through oxidative stress and immune dysfunction. Our study results not only provide a new perspective for the toxicity assessment of NH, but also enrich the toxicological mechanism of NH.