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Acrylamide (AA) has been extensively examined for its potential toxicological effects on humans and animals, but its impacts on gut microbiota and effects on hosts’ susceptibility to enteric infection remain elusive. The present study was designed to evaluate the effect of AA on gut microbiota of mice and susceptibility of mice to S. Typhimurium infection. After four weeks’ intervention, mice fed with AA exhibited significantly decreased body weight. Meanwhile, 16S rRNA gene sequencing showed reduced relative abundance of Firmicutes and increased abundance of Bacteroidetes in AA-treated mice prior to infection. In addition, we observed high relative abundance of Burkholderiales and Erysipelotrichales, more specifically the genus Sutterella and Allobaculum, respectively, in AA-treated mice before infection. Subsequently, the mice were orally infected with S. Typhimurium. The histological changes, systemic dissemination of S. Typhimurium, and inflammatory responses were examined. Compared to mice fed with normal diet, mice fed AA exhibited higher level of bacterial counts in liver, spleen, and ileum, which was consistent with exacerbated tissue damage determined by histological analyses. In addition, higher expression of pro-inflammaroty cytokines, p-IκBα, and p-P65 and lower mRNA expressions of mucin2, occludin, zo-1, claudin-1, and E-cadherin were detected in AA-treated mice. These findings provide novel insights into the potential health impact of AA consumption and the detailed mechanism for its effect on S. Typhimurium infection merit further exploration.