Dysfunction of the Hippo pathway is common in esophageal squamous carcinoma (ESCC). Chaetocin, a small molecular compound isolated from the marine fungus, exhibits potent anticancer effects. However, the anticancer effects of chaetocin on ESCC and its potential relationship to Hippo pathway remain unclear. Here, we demonstrated that chaetocin dramatically inhibited the proliferation in ESCC cells by causing cycle arrest in the M phase and activating the caspase-dependent apoptosis signaling pathwayin vitro, and we also found that chaetocin induced the accumulation cellular reactive oxygen species (ROS). The RNA-seq analysis indicated that the Hippo pathway is one of the most enriched pathways after chaetocin treatment. We further revealed that chaetocin triggered the activation of Hippo pathway in ESCC cells, which is characterized by elevated phosphorylation levels of almost all core proteins in Hippo pathway, such as MST1 (Thr183), MST2 (Thr180), MOB1 (Thr35), LAST1 (Thr1079 and Ser909) and YAP (Ser127), ultimately leading to decreased nuclear translocation of YAP. Moreover, the MST1/2 inhibitor XMU-MP-1 not only partially rescued the inhibitory effect chaetocin-induced proliferation, but also rescued the chaetocin-induced apoptosis in ESCC cells. Furthermore,in vivoresults confirmed the antitumor effect of chaetocin and its relationship with Hippo pathway. Taken together, our study demonstrates that chaetocin exhibits anticancer effects in ESCC via activation of Hippo pathway. These results provide an important basis for further research of chaetocin as a potential candidate for ESCC treatment.