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Holarrhena pubescens seeds are used in the treatment of various diseases, especially diabetes and associated complications, in different parts of the world. The present study was undertaken to determine the hypolipidemic and antihyperlipidemic effects of methanolic extract of H. pubescens seeds in rats. The extract was subjected to LC-MS analysis to determine the chemical constituents. The hypolipidemic action was studied by determining the effect of 28-day oral administration of seed extract on serum cholesterol, serum triglycerides, and serum HDL-cholesterol levels. The antihyperlipidemic action was studied in rats fed with a high-fat diet containing cholesterol and saturated fat, and the same lipid parameters were estimated during 28-day treatment. To elucidate its probable mechanism of action, in vitro studies on the inhibition of lipid accumulation in preadipocytes, DPP-IV inhibitory effect, and lipase enzyme inhibition were studied. The seed extract reduced serum levels of cholesterol and triglycerides in both normal rats and animals fed with a high-fat diet without a significant effect on HDL-cholesterol levels. The seed extract was highly effective in inhibiting lipase enzyme activity but showed a modest effect on the inhibition of lipid accumulation and DPP-IV. The results demonstrated that H. pubescens seed extract has hypolipidemic and antihyperlipidemic effects mediated probably through inhibition of lipase enzyme activity.