Salmonella enterica serotype Typhi, the etiological agent of typhoid fever, produces the Vi capsular antigen, a virulence factor absent in Salmonella enterica serotype Typhimurium. Previous studies suggest that the capsule-encoding viaB locus reduces inflammatory responses in intestinal tissue; however, there are currently no data regarding the in vivo expression of this locus. Here we implemented direct and indirect methods to localize and detect Vi antigen expression within polarized intestinal epithelial cells and in the bovine ileal mucosa. We report that tviB, a gene necessary for Vi production in S. Typhi, was significantly upregulated during invasion of intestinal epithelial cells in vitro. During infection of bovine ligated loops, tviB was expressed at levels significantly higher in calf tissue than those in the inoculum. The presence of the Vi capsular antigen was detected in calf ileal tissue via fluorescence microscopy. Together, these results support the concept that expression of the Vi capsular antigen is induced when S. Typhi transits from the intestinal lumen into the ileal mucosa.