The voltage-dependent movement, or electromotility, of cochlear outer hair cells (OHCs) contributes to cochlear amplification in mammalian hearing. Prestin, a transmembrane protein expressed in the lateral wall of the OHSs, is essential for electromotility, but molecular details of its function are unknown. Using a combined optical tweezers and whole-cell voltage clamping system, we have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) of membrane tethers, and related these parameters to prestin-associated non-linear capacitance (NLC). Our study included three test groups consisting of (i) untransfected; (ii) wild type (WT) prestin-transfected; and (iii) mutant (A100W) prestin-transfected human embryonic kidney (HEK) cells. While there was no difference in membrane effective viscosity among the three cell types, tethers from WT prestin-transfected HEK cells demonstrated altered membrane mechanical parameters and increased EMF values than control untransfected HEK cells. We found that A100W mutation in prestin, which eliminates NLC also diminishes EMF, but does not affect membrane mechanical parameters. These results suggest that prestin-associated charge transfer is associated with generation of EMF in the membrane, but independent of the effect of prestin on membrane mechanics. Based on our results, we propose synergistic effects of prestin and the membrane in the generation of NLC and electromotility. The information these results provide is important for understanding protein/membrane interactions, prestin properties and the origin of electromotility.