is a foodborne pathogen that can colonize the small intestine of the host and cause diarrhea. The alternative sigma factor RpoN plays a vital role in regulating motility, carbon utilization and affects host colonization in RIMD2210633. In this study, transcriptome and phenotypic analysis further expanded our understanding of the RpoN regulon in . A deletion mutant of (Δ ) was subjected to RNA-seq for systemic identification of the RpoN-controlled genes. Compared with the wild-type (WT), 399 genes were differentially expressed in the Δ strain. Moreover, 264 genes were down-regulated in the Δ strain, including those associated with nitrogen utilization ( ), glutamine synthetase ( ), formate dehydrogenase ( and - ), quorum sensing ( and ), polar flagellar systems, and type VI secretion system 2 (T6SS2). Quantitative real-time reverse transcription PCR (qRT-PCR) and electrophoretic mobility shift assay (EMSA) further confirmed that RpoN could directly bind to the promoters of these genes associated with polar flagellar systems ( and ), lateral flagellar systems ( and ), T6SS2 ( and ) and glutamine synthetase ( ), and then positively regulate the expression of these systems. A RpoN-binding motif was identified in using the MEME suite and verified by the EMSA. Besides, the deletion of caused a significant decrease in hemolytic activity, adhesion, and cytotoxicity. Our results provide new cues to better understand the regulatory networks of RpoN protein to motility, T6SS2, and metabolism in .