Abstract: Great progress has been achieved in the study of Hippo signaling in regulating tumorigenesis; however, the downstream molecular events that mediate this process have not been completely defined. Moreover, the regulation of Hippo signaling during tumorigenesis in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we systematically investigated the relationship between YAP-TEAD and HNF4α in the hepatocarcinogenesis of HCC cells. Our results indicated that HNF4α expression was negatively regulated by YAP1 in HCC cells via a ubiquitin proteasome pathway. By contrast, HNF4α was found to directly associate with TEAD4 to compete with YAP1 for binding to TEAD4, thus inhibiting the transcriptional activity of YAP-TEAD and the expression of their target genes. Moreover, overexpression of HNF4α was found to significantly compromise YAP-TEAD-induced HCC cell proliferation and stem cell expansion. Finally, we documented the regulatory mechanism between YAP-TEAD and HNF4α in rat and mouse tumor models, which confirmed our in vitro results. CONCLUSION: We identified a double negative feedback mechanism that controls TEAD-YAP and HNF4α expression in vitro and in vivo, thereby regulating cellular proliferation and differentiation. As YAP acts as a dominant oncogene in HCC and plays a crucial role in stem cell homeostasis and tissue regeneration, manipulating the interaction between YAP, TEADs and HNF4α may provide a new approach for HCC treatment and regenerative medicine. This article is protected by copyright. All rights reserved. 08 2016 by the American Association for the Study of Liver Diseases.