Sensation of hearing and balance is mediated by hair cells that transduce the mechanical stimulus into electrical signals. Although being subject to numerous studies the molecular nature of the transduction channel has not been revealed. In Xenopus laevis, the ion channel TRPN1 localizes to the tip of the kinocilium of inner ear hair cells, where it might be involved in mechanotransduction. TRPN1 is characterized by a large intracellular N-terminus containing 28 ankyrin repeat domains, putatively forming a molecular spring involved in the mechanotransduction process and possibly serving as binding domain for interaction partners. In this study we screened for interaction partners of the first six ankyrin repeats using a yeast-2-hybrid approach. We identified the Ca²⁺-binding protein Peflin, a member of the Penta-EF-hand protein family, as an interaction partner of TRPN1. The interaction between TRPN1 and Peflin was confirmed by GST-pull-down assays and co-immunoprecipitation experiments. In addition we found that Peflin has a distinct expression pattern when expressed heterologously in LLC-PK1-CL4 cells, a model for hair cells, showing an accumulation at the plasma membrane and an increasing accumulation over time in vesicle-like structures within the cytosol. TRPN1 on the other hand is diffusely expressed throughout the cytosol. When co-expressed, TRPN1 alters the typical membrane localisation of Peflin, supporting the biochemical interaction data. Furthermore we show in native inner ear hair cells that Peflin is located in the kinocilium of the hair bundle. Taken together our data provide evidence for an interaction of Peflin and TRPN1, suggesting that they interact in mechanotransduction.