AbstractMicrobial protein, produced by fermentation of Fusarium venenatum is a promising candidate alternative protein source. Previous study has demonstrated its ability to improve hyperlipidemia in rats, yet the related mechanism remains unclear. In this study, we aimed to evaluate the potential of F. venenatum as an alternative protein source and its impact on lipid metabolism using multi-omics analysis. Initial experiments with Caenorhabditis elegans revealed that F. venenatum enhanced longevity, improved immune responses, and reduced lipid metabolism by downregulating fat synthesis-related genes. Subsequently, we conducted experiments with mice on a high-fat diet to confirm the anti-obesity effects of F. venenatum. The groups fed F. venenatum showed improved lipid profiles and reduced hepatic fat accumulation. Furthermore, fecal metabolomic analysis showed higher excretion of primary bile acid and cholesterol in the groups fed F. venenatum which might lead to a decrease in lipid digestion and hepatic fat accumulation. Collectively, this series of experiments revealed the potential of F. venenatum as a sustainable alternative protein and its application as an anti-obesity supplement.