Mammalian hearing depends on deflection of stereocilia on the sensory outer hair cells of the inner ear. Previous data indicate that the stiffness of outer hair cell stereocilia are actively regulated. The molecular mechanism that regulate the deflection of stereocilia are presently less known. The aim of the study is to investigate the mechanistic pathway that underlie the stiffness modulation of outer hair cell stereocilia. Our hypothesis is that the membrane-cytoskeleton linker protein radixin, which is present at high concentration in stereocilia, could contribute to stiffness regulation. To test this hypothesis, we use the radixin blocker DX-52-1 which binds strongly and specifically to radixin. Time-resolved confocal imaging was used to visualize the sound-evoked motion of stereocilia in a semi-intact preparation of the guinea pig temporal bone. Cochlear microphonic potentials were also measured, using electrodes positioned in scala media. We found that the DX-52-1 inhibitor leads to an increase in stereocilia movements and decline in the amplitude of the cochlear microphonic potential. However, DX-52-1 caused a paradoxical increase in electromotility. These results suggest that radixin has a functionally important regulatory role in the mature inner ear.