In both Drosophila and mammalian systems, the Hippo pathway plays an important role in controlling organ size, mainly through its ability to regulate cell proliferation and apoptosis. The key component in the Hippo pathway is the Yes-associated protein YAP1, which localizes in nucleus, functions as a transcriptional coactivator, and regulates the expression of several proliferation- and apoptosis-related genes. The Hippo pathway negatively regulates YAP1 transcriptional activity by modulating its nuclear-cytoplasmic localization in a phosphorylation-dependent manner. Here, we describe the identification of several new PY motif-containing proteins, including angiomotin-like protein 1 (AMOTL1) and 2 (AMOTL2), as YAP1-associated proteins. We demonstrate that AMOTL1 and AMOTL2 can regulate YAP1 cytoplasm-to-nucleus translocation through direct protein-protein interaction, which can occur independent of YAP1 phosphorylation status. Moreover, down-regulation of AMOTL2 in MCF10A cells promotes epithelial-mesenchymal transition, a phenotype that is also observed in MCF10A cells with YAP1 overexpression. Together, these data support a new mechanism for YAP1 regulation, which is mediated via its direct interactions with angiomotin-like proteins.