Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified Amot family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HxRxxS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous Amot as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HxRxxS site in Amot and AmotL2 established that this site was essential for Hippo core kinases mediated phosphorylation. Wild type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted whereas Amot and Amot-S175D inhibited cell proliferation. These results collectively suggest that Hippo pathway negatively regulates actin-binding activity of Amot family members through direct phosphorylation.