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Volvariella volvacea has bioactivities in improving immunity, anti-oxidation, and alleviating obesity, which is an excellent functional food. Polysaccharide from Volvariella volvacea (VPS), one of the main bioactive components, exerts a potential fat-lowering effect, but its exact mechanism remains unclear. In this study, the effects and molecular pathways of VPS regulate the fat deposition of Caenorhabditis elegans. Results showed that VPS at low (250 μg/ml), medium (500 μg/ml) and high (750 μg/ml) concentrations all reduced the overall fat, without inhibitory effects on the growth and movement abilities of nematode. VPS at 500 μg/ml could dramatically decrease the triglyceride (TG) level of wild-type nematode, while no significant changes in TG content were observed in mutants deficient in aak-2 (energy receptor), nhr-49 (nuclear transcription factor), fat-5, and fat-7 genes. VPS declines fat storage of C. elegans, largely through the aak-2/nhr-49-mediated fatty acid synthesis pathway, and partially the acs-2-mediated fatty acid oxidation pathway. PRACTICAL APPLICATIONS: A model illustrates the mechanism of polysaccharide from Volvariella volvacea (VPS) inhibiting fat accumulation in Caenorhabditis elegans. VPS may directly or indirectly activate the energy sensor aak-2, which governs lipid metabolism. Results demonstrate that VPS regulates fat metabolism including fatty acid oxidation (FAO) and fatty acid synthesis (FAS), rather than lipolysis. In the FAO, VPS promotes FAO by up-regulating the mRNA and protein levels of acs-2. In FAS, VPS significantly down-regulated the transcriptional regulator nhr-49 and the downstream targets fat-5, fat-6, and fat-7, thereby declining the overall fat deposition. In conclusion, VPS inhibits the fat accumulation of C. elegans largely dependent on an aak-2/nhr-49-mediated FAS pathway.