While prestin-mediated outer hair cell (OHC) electromotility provides mechanical force for sound amplification in the mammalian cochlea, proper OHC stiffness is required to maintain normal electromotility and to transmit mechanical force to the basilar membrane (BM). To investigate the in vivo role of OHC stiffness in cochlear amplification, chlorpromazine (CPZ), an antipsychotic drug that alters OHC lateral wall biophysics, was infused into the cochleae in living guinea pigs. The effects of CPZ on cochlear amplification and OHC electromotility were observed by measuring acoustically and electrically-evoked BM motions. CPZ significantly reduced cochlear amplification as measured by a decline of the acoustically-evoked BM motion near the best frequency (BF) accompanied by a loss of nonlinearity and broadened tuning. It also substantially reduced electrically-evoked BM vibration near the BF and at frequencies above BF (up to 80 kHz). The high frequency notch (near 50 kHz) in the electrically-evoked BM response shifted towards higher frequency in a CPZ concentration-dependent manner with a corresponding phase change. In contrast, salicylate resulted in a shift in this notch towards lower frequency. These results indicate that CPZ reduces OHC-mediated cochlear amplification, probably via its effects on the mechanics of the OHC plasma membrane rather than via a direct effect on the OHC motor, prestin. Through modeling, we propose that with a combined OHC somatic and hair bundle forcing, the upward shift of the 50 kHz notch in the electrically evoked BM motion may indicate stiffness increase of the OHCs that is responsible for the reduced cochlear amplification.