Background: Planar cell polarity (PCP) signaling regulates the coordinated polarization of cells and is required for the normal development and function of many tissues. Previous studies have identified conserved PCP genes, such as Van Gogh-like 2 (Vangl2) and Prickle (Pk), in the regulation of coordinated orientation of inner ear hair cells and female reproductive tract development. Testin shares a PET-LIM homology with Pk. It is not clear whether Testin acts in PCP processes in mammals. Results: We identified Testin as a Vangl2-interacting protein through a 2-hybrid screen with a cochlea cDNA library. Testin is enriched to cell–cell boundaries in the presence of Vangl2 in cultured cells. Genetic inactivation of Testin leads to abnormal hair cell orientation in the vestibule and cellular patterning defects in the cochlea. In addition, Testin genetically interacts with Vangl2 to regulate hair cell orientation in the cochlea and the opening of the vaginal tract. Conclusions: Our findings suggested Testin as a gene involved in coordinated hair cell orientation in the inner ear and in female reproductive tract development. Furthermore, its genetic interaction with Vangl2 implicated it as a potential molecular link, responsible for mediating the role of Vangl2-containing membranous PCP complexes in directing morphologic polarization. Developmental Dynamics 242:1454–1465, 2013. © 2013 Wiley Periodicals, Inc.