Fenugreek (Trigonella foenum-graecum) seeds, used as a condiment, are documented for health benefits including amelioration of abnormalities in lipid homeostasis due to its hypolipidemic properties. However, molecular mechanisms underlying the hypolipidemic effect of fenugreek seeds remain obscure. In this study, hypolipidemic effect of a novel thermostable extract of fenugreek seeds (TEFS) was evaluated in vitro by employing differentiating and differentiated 3T3-L1 cells, and HepG2 cells cultured in normal or sterol-enriched conditions. Hypolipidemic effect was studied by quantifying decrease in accumulation of fat or by western blot analysis of adipogenic and lipogenic factors. At molecular level, TEFS inhibited accumulation of fat in differentiating and differentiated 3T3-L1 cells via decreased expression of adipogenic factors such as peroxisome proliferators activated-receptor-gamma (PPAR-gamma), sterol regulatory element-binding protein-1 (SREBP-1), and CAAT element-binding proteins-alpha (c/EBP-alpha). We also show that following TEFS treatment, cellular triglycerides (TGs), and cholesterol concentrations decreased significantly (P < 0.05) in HepG2 cells via reduced expression of SREBP-1, at mRNA as well as protein level. Under sterol enriched condition, TEFS upregulated low-density lipoprotein receptor (LDLR) expression resulting in enhanced LDL uptake. Treating fat supplement fed C57BL6/J mice with TEFS for 15 days resulted in decrease of serum TG, LDL-cholesterol (LDLc), and body weight in a dose- and time-dependent manner (P < 0.05). Results indicate that hypolipidemic effect of TEFS is due to inhibition of fat accumulation and upregulation of LDLR. Taken together, the study suggests that TEFS may have potential application in the management of dyslipidemia and its associated metabolic disorders.