TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. In vitro, TRIOBP isoform 4 (TRIOBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRIOBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. To examine the potential role of R1 and R2 motifs in F-actin binding, we generated TRIOBP-4 mutant proteins deleted for R1 and/or R2, and then assessed their actin-binding activity and bundle formation in vitro using actin cosedimentation assays, and fluorescence and electron microscopy. Cellular distributions of the TRIOBP-4 mutants were examined by confocal microscopy. We showed that deletion of both R1 and R2 motifs completely disrupted the actin binding/bundling activities of TRIOBP-4 and impaired its localization to cellular actin cytoskeleton structures. By contrast, TRIOBP-4, lacking only R2 motif, retained its F-actin bundling ability and remained localized to actin filaments in cells, similar to full length TRIOBP-4. On the contrary, the R1 motif-deleted TRIOBP-4 mutant, which mainly consists of the R2 motif, formed thin F-actin bundles in vitro but failed to colocalize to actin filaments in cells. These results indicate that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific.