Epithelial-mesenchymal transition (EMT) and anoikis resistance are crucial characteristics for tumor cell metastasis. High-temperature requirement A1 (HTRA1) has been identified as a serine protease with chaperone functions, but its role in the regulation of EMT, anoikis resistance, and metastasis in colorectal cancer (CRC) remains poorly understood. In this study, we identified that HTRA1 was downregulated in CRC tissues, and its low expression was significantly associated with advanced TNM stage and poor prognosis. Loss of HTRA1 facilitated EMT and anoikis resistance in CRC cells, thereby potentiating metastatic potential both in vitro and in vivo. Conversely, HTRA1 overexpression produced opposite effects. Furthermore, we carried out RNA-seq and found that HTRA1 was probably involved in the regulation of Hippo/YAP1 pathway. HTRA1 overexpression led to increased phosphorylation of YAP1 and decreased nuclear translocation, which could be largely reversed by XMU-MP-1, an inhibitor of the Hippo pathway. Mechanistically, HTRA1 directly bound to and stabilized large tumor suppressor gene 2 (LATS2), a key kinase of the Hippo pathway, which contributed to the inactivation of YAP1. Restoring LATS2 expression in HTRA1-deficient CRC cells decreased EMT and anoikis resistance. Altogether, our findings unveiled the negative regulatory function of HTRA1 in CRC progression through the regulation of the Hippo/YAP1 pathway, and supported HTRA1 as a potential therapeutic target in CRC.