Three recombinant single-chain insulin mutants, [B10Glu]Ins/IGF-1(C), [A5Glu]PIP, and [A8Tyr]PIP were prepared by means of protein engineering. In the three mutants, the residue at position 10 of insulin B chain and positions 5 and 8 of insulin A chain were substituted by the corresponding residue Glu9, Glu46, and an aromatic residue at 49 of IGF-1, respectively. The growth-promoting activity of [B18Glu]Ins/IGF-1(C) is 58-fold higher than insulin and of [A5Glu]PIP, and [A8Tyr]PIP is 2.5- and 4.7-fold higher than PIP, respectively, measured using a mouse mammary tumor cell line GR2H6 in vitro. The results show that the residues Glu9, Glu46, and Phe49, especially the Glu9, play an important role in the growth-promoting function of IGF-1. Based on the results, we suggest that the insulin could be used as a scaffold to investigate the contributions of residues of IGF-1 to its biological function.