This study explores whether Nissle 1917 (EcN) can preserve the integrity of the intestinal barrier by modulating the metabolism pathway of short-chain fatty acids (SCFAs) in a C57BL/6J mouse model of lipopolysaccharide (LPS)-induced acute enteritis and a model of a Caco-2 monolayer. The study involved establishing a septic shock model in mice through lipopolysaccharide (LPS) injection. Clinical scores and intestinal permeability were meticulously documented. Immunofluorescence was utilized to localize the tight junction proteins. A quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of G protein-coupled receptors (GPRs) signaling. Additionally, the supplement of acetate and butyrate with Caco-2 monolayers to elucidate the potential of EcN in augmenting the intestinal barrier primarily via the modulation of SCFAs and qRT-PCR was performed to detect the expression of tight junction proteins and the activation of the GPRs protein signaling pathway. EcN mitigated the clinical symptoms and reduced intestinal permeability in the colon of LPS-induced mice. It also enhanced the production of SCFAs in the gut and upregulated the expression of SCFA receptor proteins GPR41 and GPR43 in the colon tissue. Our findings reveal that EcN activates the SCFA/GPRs pathway, thereby preserving intestinal barrier function and alleviating inflammation in a mouse sepsis model.