Salmonella enterica serovar Typhi z66 positive strain contains a fljBA-like operon on a linear plasmid. The operon contains the gene fljB:z66 which encodes the z66 antigen. RpoE is a sigma factor σ(E) that initiates transcription of a series of genes in Escherichia and Salmonella under environmental stresses. To investigate whether the gene fljB:z66 is regulated by RpoE (σ(E)), a rpoE deletion mutant of S. enterica serovar Typhi (ΔrpoE) was prepared in this study. The defective motility of the ΔrpoE was confirmed firstly. Transcriptional expression of flagellar genes was screened using a genomic DNA microarray. Some class-2 and most class-3 flagellar genes were downregulated in the ΔrpoE after 30 min of hyperosmotic stress. The expression of fliA and fljB:z66, a class-2 flagellar gene and a class-3 flagellar gene, obviously decreased; however, expression of the class-1 flagellar genes flhDC did not change obviously in the ΔrpoE compared to the wild-type strain in the same conditions. Results of quantitative real-time PCR (qRT-PCR) showed that the expression levels of fliA and fljB:z66 in the ΔrpoE after 30 min of hyperosmotic stress decreased about five and eightfold, respectively, compared to the wild-type strain. Similar results were observed at 120 min of hyperosmotic stress. Western blotting and qRT-PCR analysis showed that expression of fliA and fljB:z66 was significantly increased after supplemental expression of rpoE with a recombinant plasmid pBADrpoE in the ΔrpoE strain. These results demonstrated that RpoE promoted the expression of class-3 flagellar genes and it might be performed by initiating the expression of fliA in S. enterica serovar Typhi under hyperosmotic stress.