Targeted deletion of mXinalpha results in cardiac hypertrophy and cardiomyopathy with conduction defects (Gustafson-Wagner, E., Sinn, H. W., Chen, Y.-L., Wang, D.-Z., Reiter, R. S., Lin, J. L.-C., Yang, B., Williamson, R. A., Chen, J. N., Lin, C.-I., and Lin, J. J.-C. (2007) Am. J. Physiol. 293, H2680-H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXinalpha and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXinalpha directly interacts with beta-catenin. The beta-catenin-binding site on mXinalpha was mapped to amino acids 535-636, which overlaps with the known actin-binding domains composed of the Xin repeats. The overlapping nature of these domains provides insight into the molecular mechanism for mXinalpha localization and function. Purified recombinant glutathione S-transferase- or His-tagged mXinalpha proteins are capable of binding and bundling actin filaments, as determined by co-sedimentation and electron microscopic studies. The binding to actin was saturated at an approximate stoichiometry of nine actin monomers to one mXinalpha. A stronger interaction was observed between mXinalpha C-terminal deletion and actin as compared with the interaction between full-length mXinalpha and actin. Furthermore, force expression of green fluorescent protein fused to an mXinalpha C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXinalpha. These results suggest a model whereby the C terminus of mXinalpha may prevent the full-length molecule from binding to actin, until the beta-catenin-binding domain is occupied by beta-catenin. The binding of mXinalpha to beta-catenin at the adherens junction would then facilitate actin binding. In support of this model, we found that the actin binding and bundling activity of mXinalpha was enhanced in the presence of beta-catenin.