ABSTRACTSalmonellaEnteritidis is a foodborne enteric pathogen that infects humans and animals, utilizing complex survival strategies. Bacterial small RNA (sRNA) plays an important role in these strategies. However, the virulence regulatory network ofS. Enteritidis remains largely incomplete and knowledge of gut virulence mechanisms of sRNAs is limited. Here, we characterized the function of a previously identifiedSalmonellaadhesive-associatedsRNA (SaaS) in the intestinal pathogenesis ofS. Enteritidis. We found that SaaS promoted bacterial colonization in both cecum and colon of a BALB/c mouse model; it was preferentially expressed in colon. Moreover, our results showed that SaaS enhanced damage to mucosal barrier by affecting expressions of antimicrobial products, decreasing the number of goblet cells, suppressing mucin gene expression, and eventually reducing thickness of mucus layer; it further breached below physical barrier by strengthening invasion into epithelial cells in Caco-2 cell model as well as decreasing tight junction expressions. High throughput 16S rRNA gene sequencing revealed that SaaS also altered gut homeostasis by depleting beneficial gut microbiota while increasing harmful ones. Furthermore, by employing ELISA and western blot analysis, we demonstrated that SaaS regulated intestinal inflammation through sequential activation P38-JNK-ERK MAPK signaling pathway, which enabled immune escape at primary infection stage but strengthened pathogenesis at later stage, respectively. These findings suggest that SaaS plays an essential role in the virulence ofS. Enteritidis and reveals its biological role in intestinal pathogenesis.