Fibroin, the protein of silk, and hydrolyzed fibroin have recently been described to enhance insulin sensitivity and glucose metabolism in 3T3-L1 adipocytes. Here, we report that a series of synthetic peptides derived from the fibroin sequence have enhancing effects on glucose transport in normal and insulin-resistant 3T3-L1 cells. We observed that, among several enzymatic hydrolysates of fibroin, the chymotryptic and peptic hydrolysates were significantly more effective than others in augmenting insulin-stimulated glucose uptake in both cells. We synthesized several peptides of repetitive sequences in fibroin. Treatment with synthesized hexapeptides enhanced insulin-stimulated glucose uptake more than tri-, tetra- or pentapeptides. Among those, the effect of Gly-Ala-Gly-Ala-Gly-Tyr (GAGAGY) was most robust, and especially its activity of blocking off the chronic-insulin-induced loss of insulin-stimulated uptake was remarkable. Data reveal that the residues of tyrosine situated at the ends of the peptides play a critical role for exerting their activities. We demonstrate that the insulin-sensitizing effect of GAGAGY is due to enhancement of phosphoinositide 3-kinase (PI 3-K) signaling pathway. The GAGAGY-induced insulin-stimulated glucose uptake was sensitive to inhibition of PI 3-K by wortmannin. Phosphorylation of Akt was also elevated in GAGAGY-treated cells. Furthermore, GAGAGY significantly increased insulin-induced glucose transporter 4 (GLUT4) translocation without affecting the synthesis of GLUT4. Our findings suggest that fibroin-derived peptides such as GAGAGY could be considered as novel insulin-sensitizing agents with an activity of blocking the development of insulin resistance.