Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The non-linear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, whose precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a FRET-based technique. We heterologously expressed prestin labeled with fluorophores at the C- or N-terminus in HEK 293T cells, and monitored FRET changes upon depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the C- termini and between the C-and N-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish non-linear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the C-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements.