AbstractMyoD is a DNA‐binding protein capable of specific interactions that involve the helix–loop–helix (HLH) domain. The HLH motif of MyoD can form oligomers with the HLH motif of Id1 (the inhibitor of DNA‐binding proteins) that folds into a highly stable helical conformation stabilized by the self‐association. The Id family consists of four related proteins that contain a highly conserved dimerization motif known as the HLH domain. In signaling pathways, Id proteins act as dominant negative antagonists of the basic helix–loop–helix (bHLH) family of transcription factors which play important roles in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by binding as dominant negative HLH proteins to form high‐affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation‐associated genes. The goal of this study is to design and synthesize peptide fragments of MyoD with high affinity for Id1 to interrupt the interactions among Id1, MyoD, and other bHLH DNA‐binding proteins and to inhibit the proliferation of cancer cells. Affinity of each peptide for Id1 was determined by surface plasmon resonance (SPR) technology. The secondary structure of each peptide was studied by circular dichroism (CD) spectroscopy. Biological effects of each peptide in several cancer cells such as breast and colon cancer cells were analyzed. Results demonstrated that the peptide 3C (H‐Tyr‐Ile‐Glu‐Gly‐Leu‐Gln‐Ala‐Leu‐Leu‐Arg‐Asp‐Gln‐NH2) not only showed high affinity for Id1 but also exhibited antiproliferative effects in HT‐29 and MCF‐7 cancer cells; the IC50 value of 3C was determined as 25 µM in both cells. The percentage of sub‐G1 in the cell cycle of the cancer cells treated with 5 µM of 3C was increased, indicating the induced apoptosis of cancer cells by 3C. Taken together, the peptide 3C is a promising lead compound for the development of antiproliferative agents. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.