Salmonella enterica serovar Typhi is a human enteroinvasive pathogen that can overcome the stress caused by the high osmolarity of the human small intestine and cause systemic infection. To investigate the global transcriptional regulations of S. enterica serovar Typhi exposed to a hyperosmotic environment, a genomic oligo-DNA microarray containing 4474 Salmonella genes was prepared. A wild strain of S. enterica serovar Typhi GIFU10007 was grown in LB medium containing 50 mM NaCl to simulate a low osmotic environment. The hyperosmotic stress was simulated by an osmotic up-shift, which increased the concentration of NaCl in the LB from 50 mM to 300 mM. Genome-wide gene expressions of S. enterica serovar Typhi at 15 min, 30 min, 60 min, and 120 min after the osmotic up-shift were investigated by the microarray analysis. Gene expression profiles in somewhat later stage (60 similar to 120 min) of the stress were quite different from those in the early stage (0 similar to 30 min) of the stress. At 120 min after the osmotic stress, the expression levels of 889 genes were obviously changed. However, expression levels of only 382 genes were significantly changed at 15 min after the osmotic stress. The expression levels of most SPI-1 genes associated with invasion of the pathogen were increased at 120 min after the osmotic up-shift, but were not obviously changed at 15 min or 30 min after the osmotic stress. Expressions of a central regulatory gene, phoP, and sigma factor genes rpoE, rpoD, and rpoS were also changed with different profiles during the osmotic stress. These results indicated that the invasive ability of the pathogen is significantly increased after 2 h of hyperosmotic stress, and regulator PhoP and sigma factors RpoE, RpoD appear to participate in the network regulatory mechanisms that benefit the pathogen to adapt hyperosmotic environmental conditions. The later increased invasive ability of S. enterica serovar Typhi after hyperosmotic stress may be one reason why the pathogen performs invading in the distal ileum of human and not in areas of the upper small intestine.