During the infectious procedure of Salmonella enterica serovar Typhi (S. Typhi), Salmonella would suffer from some severe environmental stresses, such as gastric acid stress, enteric hyperosmotic stress, bile acids stress, and oxidative stress. S. Typhi must regulate the expression of numerous genes through the complex regulatory network to adapt strict stresses. RpoS, which encodes sigma factor sigma(s), was reported to be a very important regulator in the maximal survival of enteric pathogens including S. enterica in the stress conditions. However, the role of RpoS in S. Typhi under early hyperosmotic stress is not clear. In this study, we prepared the RpoS-deleted mutant (a-(3)RpoS) and compared the growth of a-(3)RpoS and wild-type strain. In addition, we analyzed its global transcription profile under early hyperosmotic stress by a microarray. The results showed that a-(3)RpoS grew significantly slower than wild-type strain and 24 genes displayed differential expression between the wild-type strain and Delta RpoS upon 30-min exposure in the high osmolarity. Most of these genes are associated with enzymes of metabolism. Taken together, our study firstly demonstrated that RpoS affects gene expression in S. Typhi under early hyperosmotic stress and may impact the growth of bacteria by regulating bacterial metabolic enzymes.