Intestinal perforations lead to a high risk of sepsis-associated morbidity and multi-organ dysfunctions. A perforation allows intestinal contents (IC) to enter the peritoneal cavity, causing abdominal infections. Right- and left-sided perforations have different prognoses in humans, but the mechanisms associated with different cecum and colon perforations remain unclear. This study investigates how gut flora influences outcomes from perforations at different sites in mice. Using fecal-induced peritonitis mouse model, isolated IC from the cecum or colon was injected peritoneally at 2 mg/kg. Bacterial burden was quantified with quantitative PCR, and microbial communities were analyzed using 16S rRNA gene sequencing. Survival rates were monitored, and blood biochemical indices, histological changes, cytokines expression, immunological signaling and multiple-organ damage were assessed at 16 h post-injections. The results showed cecum IC developed more severe sepsis than colon IC, with shorter median survival time and greater multi-organ damage. Mice treated with cecum IC displayed elevated tissue damage markers in the liver, heart, and kidneys, contributing to worsened pathology. This was likely driven by systematic inflammatory cytokines production and lung inflammation. Mechanistically, cecum IC triggered stronger cGAS-STING and TBK1-NF-κB signaling, promoting systemic inflammation compared to the colon IC. Moreover, bacterial analysis demonstrated that cecum IC carry a higher bacterial burden than colon IC and exhibit a different microbial community. A detailed microbiome comparison revealed an increased abundance of potentially pathogenic bacteria in the cecum IC. These findings suggest that the site of intestinal perforation influences sepsis severity, with the cecum being associated with a higher bacterial burden and a relatively increased abundance of potentially pathogenic bacteria compared to the colon. Our findings first compared the lethality associated with the microbial composition of the cecum and colon, indicating the perforation site could help providers predict the severity of sepsis, thereby introducing a novel perspective to microbiology and sepsis research.Copyright © 2025 Elsevier B.V. All rights reserved.