Macroalgae Laminaria japonica (MLJ) has been reported to exhibit various biological activities including improving immunity, anti-aging, anti-tumor, anti-atherosclerosis and anti-diabetic, but the protective mechanisms of MLJ consumption against non-alcoholic fatty liver disease (NAFLD) associated with hyperlipidemia remain poorly understood. This study demonstrated that MLJ consumption prevented high-fat diet (HFD)-induced NAFLD associated with hyperlipidemia in a rat model, and improved hyperlipidemia-related parameters, e.g. serum and hepatic lipid profiles. Moreover, histological analysis showed that MLJ reduced lipid deposition in adipocytes and hepatocytes compared with the HFD group. Such beneficial effects may be associated with the modulation of the intestinal microbiota, especially some key microbial phylotypes involved in lipid metabolism homeostasis. The underlying protective mechanisms of MLJ consumption against HFD-induced NAFLD associated with hyperlipidemia were also studied by ultra-high performance liquid chromatography with quadruple-time of flight mass spectrometry (UPLC-QTOF/MS)-based liver metabolomics coupled with pathway analysis. The metabolic pathway enrichment analysis of the differentially abundant hepatic metabolites indicated that primary bile acid biosynthesis metabolism and cysteine and methionine metabolism were the two main metabolic pathways altered by MLJ consumption when compared with the model group. The analysis of the transcription levels of liver-related genes by RT-qPCR and the expressions of liver-related proteins by immunohistochemistry (IHC) showed that MLJ consumption could regulate the levels of mRNA transcription and protein expression related to hepatic lipid metabolism. In short, this study indicates that MLJ could be developed as functional food supplement for the prevention or treatment of NAFLD associated with hyperlipidemia.