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 (GB) through biofilms on gallstones, additional anatomic sites have been suggested that could also harbor Salmonella. S. Typhimurium, orally infected 129 × 1/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 days 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 adaptative niche for Salmonella spp. aggregates/biofilms and is a putative site for long-term infection.IMPORTANCETyphoid fever is a systemic infectious disease triggered by the gastrointestinal dissemination of Salmonella Typhi and Paratyphi in humans. Three to five percent of infected individuals become chronic carriers, a state in which gallstone biofilm formation facilitates spread of the bacteria in feces. Notably, surgical removal of the gallbladder (GB) in some chronic carriers (22%) does not guarantee the elimination of the bacteria, and the rationale for this remains poorly understood. This study is significant as it explores other tissues associated with the chronic carrier state. It highlights not only a cholesterol-rich diet as an important etiological factor for Salmonella colonization but also identifies the cecum as a crucial tissue promoting fecal shedding. Additionally, we determined that biofilm matrix components of Salmonella are key factors contributing to these effects. A greater understanding of these mechanisms will allow the formulation of new therapeutic strategies specifically targeted at preventing typhoid fever dissemination from chronic carriers.