Salmonella enterica serovar Enteritidis is a common foodborne pathogen that infects both humans and animals. The S. Enteritidis virulence regulation network remains largely incomplete, and knowledge regarding the specific virulence phenotype of small RNAs (sRNAs) is limited. Here, we investigated the role of a previously identified sRNA, Salmonella adhesive-associated sRNA (SaaS), in the virulence phenotype of S. Enteritidis by constructing mutant (ΔsaaS) and complemented (ΔsaaS/psaaS) strains. SaaS did not affect S. Enteritidis; it was activated in the simulated intestinal environment (SIE), regulating the expression of virulence target genes. We discovered that it directly binds ssaV mRNA. Caco-2 and RAW 264.7 cell assays revealed that SaaS promoted S. Enteritidis invasion and damage to epithelial cells while suppressing macrophage overgrowth and destruction. Furthermore, a BALB/c mouse model demonstrated that the deletion of SaaS significantly reduced mortality and attenuated the deterioration of pathophysiology, bacterial dissemination into systemic circulation, and systemic inflammation. Our findings indicate that SaaS is required for S. Enteritidis virulence and further highlight its biological role in bacterial pathogenesis. IMPORTANCE Salmonella is a zoonotic pathogen with high virulence worldwide, and sRNAs have recently been discovered to play important roles. We explored the biological characteristics of the sRNA SaaS and developed two cell infection models and a mouse infection model. SaaS is an SIE-responsive sRNA that regulates the expression of virulence-targeted genes. Additionally, it differentially mediates invasion and intracellular growth for survival and infection of the epithelium and macrophages. We further found that SaaS enhanced bacterial virulence by promoting lethality, colonization, and inflammatory response. These findings provide a better understanding of the critical role of sRNA in bacterial virulence.