ABSTRACT Hippo signaling pathway is emerging as a novel target for anticancer therapy because it plays key roles in organ size control and tumorigenesis. As the downstream effectors, Yes‐associated protein (YAP)‐transcriptional enhancer activation domain family member (TEAD) association is essential for YAP‐driven oncogenic activity, while TEAD is largely dispensable for normal tissue growth. We present the design of YAP‐like peptides (17mer) to occupy the interface 3 on TEAD. Introducing cysteines at YAP sites 87 and 96 can induce disulfide formation, as confirmed by crystallography. The engineered peptide significantly improves the potency in disrupting YAP‐TEAD interaction in vitro . To confirm that blocking YAP‐TEAD complex formation by directly targeting on TEAD is a valid approach, we report a significant reduction in tumor growth rate in a hepatocellular carcinoma xenograft model after introducing the dominant‐negative mutation (Y406H) of TEAD1 to abolish YAP‐TEAD interaction. Our results suggest that targeting TEAD is a promising strategy against YAP‐induced oncogenesis.—Zhou, Z., Hu, T., Xu, Z., Lin, Z., Zhang, Z., Feng, T., Zhu, L., Rong, Y., Shen, H., Luk, J. M., Zhang, X., Qin, N. Targeting Hippo pathway by specific interruption of YAP‐TEAD interaction using cyclic YAP‐like peptides. FASEB J . 29, 724‐732 (2015). www.fasebj.org