Salmonella enterica serovar Enteritidis is a primary pathogen causing foodborne diseases and intestinal inflammatory responses. Acid tolerance response (ATR), as a strategy of adaption and resistance to acid stress, may contribute to enhanced virulence. In this study, there was a moderately acid adaption (pH 5.0) for S. Enteritidis cells prior to treatment with acid stress (pH 3.0). To figure out whether S. Enteritidis up-regulated the virulence or not, a global transcriptomic analysis was carried out by high-throughout RNA-sEq. The results showed 74 differentially expressed genes (DEGs) involved in virulence were identified after acid stress, among which, 62 DEGs were up-regulated and 12 DEGs were down-regulated. Afterwards, those virulence-linked DEGs were discussed and classified into four aspects based on the steps of infection, including flagellar functions, fimbrial adhesins, T3SS-mediated invasion and other virulent determinants. In conclusion, S. Enteritidis seemed to exhibit a trend of virulent genes towards high-expression under acid stress, revealing risks of Salmonella in acid-containing food. To our knowledge, there were few studies on comprehensively analyzing virulent genes expression changes of Salmonella, but it’s novel to put forward pathogenicity as the highest priority under acid environment.