Edible mushrooms have now been suggested as promising sources of biological functional ingredients and are the subject of the most recent nutrition research and novel functional foods. Polysaccharides from mushrooms exhibit impressive biological effects, notably against obesity. Obesity is a chronic metabolic disorder characterized by chronic inflammation, gut dysbiosis, and hyperpermeability of the colon. Here, we prove that mushrooms Morchella esculenta polysaccharide (MEP) effects on HFD-induced obesity, colonic inflammation, and gut microbiota dysbiosis. Our findings demonstrate MEP supplementation attenuates obesity parameters and reduces inflammation in the colon via regulation of Toll-like receptor 4 (TLR4), nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inactivation of nuclear factor kappa B (NF-κB). Furthermore, MEP administration restores gut microbiota dysregulation by ameliorating Firmicutes to Bacteroidetes proportion as well as enhancing beneficial bacteria, like Lactobacillus, and inhibiting pathogenic bacteria like Enterococcus. MEP improves gut integrity by increasing tight junction proteins (TJs) and reducing endotoxin levels by controlling Lipopolysaccharide (LPS) in HFD-induced obese mice. These results demonstrated the therapeutic efficacy of MEP in attenuating HFD-induced obesity via regulating inflammatory cascades, ameliorating the gut microbiome, and modulating gut integrity.