Active compounds with antidiabetic potential were isolated from silk peptide E5K6 by consecutive ultrafiltration and gel filtration using Biogel P-2 and RS-HPLC using a YMC-Pack Pro C18 column. The highest α-glucosidase inhibitory activity of silk peptide E5K6 resulted from fractions with MW <1 kDa. The activities of gel-filtered fractions from silk peptide E5K6 of <1 kDa were assayed in vitro, demonstrating that the fourth peak (F4) had the highest α-glucosidase inhibitory activity (IC(50) = 37.1 mg/mL). F4 of silk peptide E5K6 was separated by HPLC into two peaks. Moreover, the purified compounds were identified as Gly-Glu-Tyr (GEY, MW = 367 Da) and Gly-Tyr-Gly (GYG, MW = 295 Da) according to amino acid sequences, and their α-glucosidase inhibitory activities (IC(50)) were 2.7 and 1.5 mg/mL, respectively.