Irrational use of antibiotics against Gram-negative bacteria results not only in gut dysbiosis but also leads to antimicrobial resistance (AMR). Consequently, natural products are being considered as an alternative to antibiotics. One such product is Lentinan (LNT), a polysaccharide found in edible mushroom, Lentinula edodes. This study was performed to evaluate the effects of LNT on overall intestinal health and gut microbiota in mice exposed to LPS. In this study, 30 male ICR mice were randomly divided into Control (C), Model (M), and Prophylactic (P) groups. For 13 days, groups C and M received a basal diet + normal saline, while group P received a basal diet + LNT (200 mg/kg of BW). Then groups M and P were exposed to LPS intraperitoneal injection (IP) (10 mg/kg) for 9 hours, and all mice were sacrificed for analysis on day 14^th. LNT treatment significantly increased levels of glutathione peroxidase (GSH:Px) (P<0.05), interleukin-10 (IL-10) (P<0.0001), increased villi height (P<0.0001), the crypt depth (P<0.001), and beneficially modulated the relative abundance of bacteria, especially phylum Firmicutes, Bacteroidetes, Actinobacteria and genus Campylobacter. Other genera Duncaniella (P<0.001), CAG-269 (P<0.05), and Tidjanibacter (P<0.05) were increased, while Phocaeicola_A_858004 (P<0.05), Enterococcus (P<0.0001) and D16-34 (P<0.05) were decreased after LNT supplementation. Altogether, LNT treatment improved antioxidant enzyme activity, intestinal integrity, and immune response, reduced inflammation, and induced a beneficial shift in gut microbiota to mitigate LPS-induced sepsis.