In this study, the hypolipidemic effects of Tamarindus indica fruit pulp extract (Tifpe) in the serum obtained from diet-induced hypercholesterolemic hamsters were investigated using two-dimensional gel electrophoresis and mass spectrometry. Significantly altered levels of serum proteins were validated using ELISA. Hamsters fed with Tifpe, both in the absence and presence of high-cholesterol diet, were shown to have significantly reduced levels of serum triglyceride, LDL-C and total cholesterol. The Tifpe fed non-hypercholesterolemic hamsters also showed significant enhanced levels of serum apolipoprotein A1 (APOA1), antithrombin III (ATIII), transferrin (TF), transthyretin and vitamin D binding protein (VDBP). In diet-induced hypercholesterolemic hamsters, APOA1, ATIII and TF, which were relatively low in levels, became significantly enhanced when the hamsters were fed with Tifpe. These Tifpe-fed hypercholesterolemic hamsters also showed significant higher levels of serum VDBP. Ingenuity Pathway Analysis of the Tifpe modulated serum proteins singled out “Lipid metabolism, molecular transport, small molecule biochemistry” as the top network. Our results suggest that the hypolipidemic effects of Tifpe are associated with alterations of serum proteins that are known to be cardioprotective and involved in the metabolism of lipids.