In vitro α-glucosidase inhibitory activity of Ginkgo biloba leaves was investigated. The inhibitory activity of methanol extracts from yellow and green leaves was 13.8 and 40.1 μg mL(-1), respectively. Each methanol extract was separated into its respective fraction by solvent-solvent extraction with n-hexane, chloroform, ethyl acetate and n-butanol. The n-hexane fractions (in both methanol extracts from green and yellow leaves) exhibited high α-glucosidase inhibitory activity with IC50 values of 13.6 and 13.4 μg mL(-1), respectively. Further fractionation of the n-hexane fractions by silica gel column chromatography gave the most active fraction which was identified as ginkgolic acid (C13:0) and a mixture (C13:0, C15:0, C15: 1, C17:1 and C17:2). Ginkgolic acid (C13:0) exhibited the highest α-glucosidase inhibitory activity. This is the first study to successfully isolate ginkgolic acids as α-glucosidase inhibitors.