Asymptomatic chronic carriers occur in approximately 5% of humans infected with Salmonella enterica serovar Typhi (S. Typhi) and represent a critical reservoir for bacterial dissemination. While chronic carriage primarily occurs in the gallbladder through biofilms on gallstones, additional anatomic sites have been suggested that could also harbor Salmonella. S. Typhimurium, orally-infected 129X1/SvJ mice were pre-treated with a cholesterol-rich diet as a gallstone model for chronic carriage. We observed S. Typhimurium in feces and the cecum during early and persistent infection. Furthermore, bacterial biofilm-like aggregates were associated with the cecum epithelium at 7- and 21-day post-infection (DPI) in mice on a lithogenic diet (Ld) and correlated with an increase in cecal cholesterol at 21 DPI. Salmonella’s extracellular matrix (ECM) was demonstrated as important in colonizing the cecum, as survival and aggregate formation significantly decreased when mice were infected with a quadruple ECM mutant strain. Gallbladder Salmonella counts were low at 36 DPI while cecal Salmonella were high, suggesting that gallbladder colonization was likely not responsible for the high cecal burden. All cecum phenotypes were significantly diminished in mice fed a normal diet (Nd). Finally, we examined the capability of S. Typhi to colonize the cecum and showed S. Typhi in feces and in aggregates in the cecum up to 7 DPI, with slightly higher counts in mice fed a Ld compared to Nd. Our findings suggest that the cecum, particularly under cholesterol-rich conditions, serves as an adaptive niche for Salmonella spp. aggregates/biofilms and is a putative site for long-term infection