TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. TRIOBP has three isoforms, named TRIOBP-1, TRIOBP-4, and TRIOBP-5. 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. Recent our study demonstrated that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific. Structural analysis of TRIOBP by amino acid sequence showed ID proteins. Thus the second structure of TRIOBP may not have. To investigate the structural property of TRIOBP-4, we analyzed the structure of TRIOBP-4 by using circular dichroism, dynamic light scattering, and fluorescence correlation spectroscopy. Our analysis show that TRIOBP has a beta-sheet, but not alpha-helix. To investigate the structure of tight F-actin bundle structure with TRIOBP, we analyzed 3-D structure of the bundle by using 3-D image analysis from transmitted electron microscope images.