Hesperidin, abundant in citrus fruits, has a wide range of pharmacological effects, including anticarcinogenic, anti-inflammatory, antioxidative, radioprotective, and antiviral activities. However, relatively few studies on the effects of hesperidin on lipid metabolism have been reported. Here, using Caenorhaditis elegans as a model animal, we found that 100 μM hesperidin significantly decreased fat accumulation in both high-fat worms cultured in nematode growth medium containing 10 mM glucose (83.5 ± 1.2% versus control by Sudan Black B staining and 87.6 ± 2.0% versus control by Oil Red O staining; p < 0.001) and daf-2 mutant worms (87.8 ± 1.4% versus control by Oil Red O staining; p < 0.001). Furthermore, 50 μM hesperidin decreased the ratio of oleic acid/stearic acid (C18:1Δ9/C18:0) (p < 0.05), and supplementation of oleic acid could restore the inhibitory effect of hesperidin on fat accumulation. Hesperidin significantly downregulated the expression of stearoyl-CoA desaturase, fat-6, and fat-7 (p < 0.05), and mutation of fat-6 and fat-7 reversed fat accumulation inhibited by hesperidin. In addition, hesperidin decreased the expression of other genes involved in lipid metabolism, including pod-2, mdt-15, acs-2, and kat-1 (p < 0.05). These results suggested that hesperidin reduced fat accumulation by affecting several lipid metabolism pathways, such as fat-6 and fat-7. This study provided new insights into elucidating the mechanism underlying the regulation of lipid metabolism by hesperidin.