In this study, the effect of rutin on fatty acid metabolism and antioxidant activity were evaluated. We found that the antioxidant capacity of rutin-treated Caenorhabditis elegans was enhanced but the triglyceride content was significantly reduced. The reduction of fat accumulation by rutin was also confirmed by Oil Red O staining. RNA-seq analysis indicated that rutin significantly regulated the expression of seven genes related to lipid metabolism in C. elegans. Among the seven genes, acox-1.3, stdh-3, and fat-7 were associated with fatty acid metabolism. Rutin significantly reduced fat accumulation in both fat-6 and fat-7 mutant strains but did not affect the fat storage of fat-6/fat-7 double mutant, which indicated that the impact of rutin on fat storage depended on fat-6 and fat-7. These findings indicated that rutin reduced fat storage depending on the regulation of lipid metabolism-related genes expression and thereby regulating the biosynthesis of the corresponding unsaturated fatty acid.