To investigate the prebiotic potential of two Laminaria japonica polysaccharide (LJP) fractions with different molecular weights and structures, we conducted in vitro simulated digestion and fermentation with hyperlipidemia-associated human gut microbiota. The results indicated that the LJP fraction with higher molecular weight (HLJP) appeared to have a more complex monosaccharide composition and microstructure than did the LJP fraction with lower molecular weight (LLJP), and both fractions could not be digested by in vitro simulated digestion. After in vitro fermentation, HLJP generated more short-chain fatty acids (SCFAs) and showed stronger ability to regulate core metabolites. Intriguingly, LLJP is better at promoting the proliferation of Akkermansiaceae, while HLJP is more effective in reducing the Firmicutes/Bacteroidetes ratio and increasing the content of Bacteroidaceae and Tannerellaceae. The present study indicates that LLJP and HLJP may have probiotic effects through different approaches and these differences may be related to the molecular weight and structure of the polysaccharides.