Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. Gut microbiota and its metabolites alteration are closely related to NAFLD. Nootkatone is an edible flavorants derived from grapefruit which has a variety of biological activities. However, the precise mechanisms of nootkatone on NAFLD remains to be defined. Our results showed that nootkatone prevented body weight gain and decreased serum lipid level, hepatic lipogenesis, hepatic proinflammatory cytokines secretion in NAFLD mice. Also, nootkatone attenuated inflammatory response via inhibiting TLR4/NF-κB/NLRP3 pathway. Moreover, nootkatone restored intestinal barrier damage through increasing tight junction proteins and SCFAs contents. Further 16S rRNA sequencing of colonic content suggested that nootkatone recovered the disturbed gut microbiota to improve NAFLD. Spearman correlation analysis between gut microbiota and NAFLD related parameters indicated that nootkatone regulated lipid metabolism and immunity via altering the gut microbiota. In conclusion, these findings revealed that nootkatone alleviated hepatic lipid homeostasis and inflammatory response in NAFLD mice, which associated with intestinal barrier integrity and the regulation of gut microbiota. This study provides new perspectives that nootkatone has efficacy on NAFLD via “gut-liver axis”, and nootkatone is expected to be developed as a functional food additive.