Salicylate, a common drug frequently used long term in the clinic, is well known for causing reversible hearing loss and tinnitus. Our previous study, however, demonstrated that chronic administration of salicylate progressively raised the amplitude of distortion product of otoacoustic emissions (DPOAEs), which are mainly caused by (outer hair cell) OHC electromotility. How salicylate affects OHC electromotility to cause this paradoxical increase remains unclear. One possibility is that it could affect prestin, which is a motor protein that contributes to the mechano-electrical properties of OHCs. In this experiment, we assessed the effect of acute and chronic salicylate treatment on prestin expression. Interestingly, after long-term salicylate injection (200 mg/kg, twice daily for 14 days), prestin gene and protein levels were up-regulated about twofold. These levels returned to baseline 14 days after treatment stopped. Acute injection of salicylate (single injection, 400 mg/kg) did not affect prestin levels. These data reveal that chronic salicylate administration markedly, but reversibly, increased prestin levels which may contribute to the enhanced DPOAE amplitudes we observed previously with similar salicylate treatment, which may be responsible for salicylate-induced tinnitus generation.