As a popular traditional fermented beverage, kombucha has been extensively studied for its health benefits. However, the science behind the anti-inflammatory effect of kombucha has not been well studied, and there is an urgent need to uncover the secrets of the anti-inflammatory properties of kombucha. Here, we investigate kombucha's protective effects against lipopolysaccharide (LPS)-induced sepsis and on the intestinal microecology in mice. The contents of reducing sugars, polyphenols, catechins, and organic acids in the kombucha group were identified using various methods. The results showed that the concentrations of acetic acid, gluconic acid, polyphenol, and glucuronic acid in the kombucha group were 55.70 ± 2.57 g L-1, 50.20 ± 1.92 g L-1, 2.36 ± 0.31, and 1.39 ± 0.22 g L-1, respectively. The result also demonstrated that kombucha effectively improves the survival rate from 0% to 40%, and increases the thermoregulation in LPS-treated mice, which showed decreased mobility and had lost their appetite for food. Furthermore, kombucha reduced the levels of tumor necrosis factor-α and interleukins (IL)-1β and IL-6, restored the levels of T cells and macrophages in LPS-challenged mice, alleviated the histopathological damage, and inhibited NF-κB signaling in mice with LPS-induced sepsis. We demonstrated that kombucha effectively prevents cellular immune function disorder in mice at the initial stage of sepsis and exerts an immunomodulatory effect. In addition, the effect of kombucha on the gut microbiota was investigated during sepsis. Kombucha supplementation altered the diversity of the gut microbiota and promoted the growth of butyrate-producing bacteria, which exert anti-inflammatory effects. Our results illustrate the potential of kombucha as a novel anti-inflammatory agent against the development of systemic inflammatory responses associated with sepsis.