Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, a food-borne disease that is prevalent worldwide, most particularly in developing countries. RNA polymerase sigma factors RpoE (σ(E)) and RpoS (σ(S)) govern transcription initiation of two sets of genes in Escherichia and Salmonella. It was previously suggested that some genes might be coregulated by RpoE and RpoS in Salmonella under conditions of environmental stress, but experimental evidence has been lacking. We therefore constructed rpoS deletion (ΔrpoS) and double rpoE/rpoS deletion (ΔrpoE/ΔrpoS) mutants of S. Typhi and compared their growth properties with an rpoE mutant (ΔrpoE) and wild-type strains under conditions of hyperosmotic stress. We report that the ΔrpoE, ΔrpoS, and ΔrpoE/ΔrpoS strains grew more slowly under hyperosmotic stress conditions than the wild-type strain, and the ΔrpoE/ΔrpoS strain grew most slowly. The global transcriptional profiles of ΔrpoE, ΔrpoS, ΔrpoE/ΔrpoS after 30 min of hyperosmotic stress were investigated using a Salmonella genomic DNA microarray. The results of microarray indicated that the expression levels of 38 genes were markedly reduced during hyperosmotic stress in the double mutant ΔrpoE/ΔrpoS strain, but expression levels were not significantly affected by single ΔrpoE or ΔrpoS mutations. This was confirmed for several key genes by qRT-PCR. This study therefore indicated crosstalk between sigma factors RpoE and RpoS in S. Typhi under hyperosmotic conditions and provides new insights into the regulatory networks of S. Typhi.