In a previous study, we found interaction of gymnemic acid (GA) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis. We now examined interaction of GA with glycolytic and related enzymes. We found that (1) GA induced a band smearing of glycerol-3-phosphate dehydrogenase (G3PDH) as well as that of GAPDH in SDS-PAGE, (2) GA diminished the G3PDH band detected by an antibody to phosphoserine, and (3) GA inhibited the G3PDH activity. The GA-induced smearing of the G3PDH band was diminished by prior incubation of GA with γ-cyclodextrin. GA gave no effects on the electrophoretic and phosphoserine bands of other glycolytic enzymes. NAD and NADH diminished the GA-induced smearing of the G3PDH and GAPDH bands in different concentration-dependent manner. Pretreatment of G3PDH with heated SDS-containing buffer or pretreatment with hydroxylamine diminished the GA-induced smearing of G3PDH. Deacylation of GA by alkaline hydrolysis diminished the smearing of G3PDH band, thereby indicating that the acyl moieties of GA were necessary for the GA-induced smearing of G3PDH. These results indicated the interaction of GA with G3PDH, an enzyme involved in glycerol metabolism. These studies suggest that GA may have some pharmacological activities including antidiabetic activity and lipid lowering effects via interaction with GAPDH and G3PDH.