The aim of this study was to determine the effect of HDAC6 inhibition using the selective inhibitor Tubastatin A (TubA) on the regulation of tert-butyl hydroperoxide (TBHP)-treated chondrocytes and a mouse OA model. Using conventional molecular biology methods, our results showed that the level of HDAC6 increases both in the cartilage of osteoarthritis (OA) mice and TBHP-treated chondrocytes in vitro. TubA treatment effectively inhibits the expression of HDAC6, attenuates oxidative stress, reduces the level of apoptotic proteins to maintain chondrocyte survival, and suppresses the extracellular matrix (ECM) degradation. In addition, our results also revealed that HDAC6 inhibition by TubA activates autophagy in chondrocytes, whereas the protective effects of TubA were abolished by autophagy inhibitor intervention. Subsequently, the positive effects of HDAC6 inhibition by TubA were also found in a mouse OA model. Therefore, our study provide evidence that HDAC6 inhibition prevents OA development, and HDAC6 could be applied as a potential therapeutic target for OA management.