Intake of soy isoflavones is inversely associated with the risk of esophageal cancer. Numerous experimental results have supported the anticancer activity of soy isoflavones. This study aimed to determine the anti-esophageal cancer activity of 6,7,40-trihydroxyisoflavone (6,7,40-THIF), a major metabolite of daidzein, which is readily metabolized in the human body. Notably, 6,7,40-THIF inhibited proliferation and increased apoptosis of esophageal cancer cells. On the basis of a virtual screening analysis, Pin1 was identified as a target protein of 6,7,40-THIF. Pull-down assay results using 6,7,40-THIF Sepharose 4B beads showed a direct interaction between 6,7,40-THIF and the Pin1 protein. Pin1 is a critical therapeutic and preventive target in esophageal cancer because of its positive regulation of b-catenin and cyclin D1. The 6,7,40-THIF compound simultaneously reduced Pin1 isomerase activity and the downstream activation targets of Pin1. The specific inhibitory activity of 6,7,40-THIF was analyzed using Neu/Pin1 wild-type (WT) and Neu/Pin1 knockout (KO) MEFs. 6,7,40-THIF effected Neu/Pin1 WT MEFs, but not Neu/Pin1 KO MEFs. Furthermore, the results of a xenograft assay using Neu/Pin1 WT and KO MEFs were similar to those obtained from the in vitro assay. Overall, we found that 6,7,40-THIF specifically reduced Pin1 activity in esophageal cancer models. Importantly, 6,7,40-THIF directly bound to Pin1 but not FKBP or cyclophilin A, the same family of proteins. Because Pin1 acts like an oncogene by modulating various carcinogenesis-related proteins, this study might at least partially explain the underlying mechanism(s) of the anti-esophageal cancer effects of soy isoflavones.