Osteoarthritis (OA) is involved in these pathophysiological changes of articular cartilage, subchondral bone and synovium. As a selective HDAC6 inhibitor, Ricolinostat (ACY-1215) has demonstrated chondroprotective effects in OA. However, its efficacy remains unclear in subchondral bone. In this study, we found that the mRNA and protein levels of HDAC6 were elevated in human OA osteoblasts in vitro. PI3K/AKT signaling pathway was suppressed with downregulation of VEGF expression in osteoblasts after ACY-1215 treatment. ACY-1215 promoted apoptosis of OA osteoblast in a concentration-dependent manner, and the expression of apoptosis-related proteins was also changed by activating caspase pathway. Moreover, western blotting showed decreased expression of MMP9 and MMP13 in IL-1β-induced chondrocytes after co-culture with ACY-1215-stimulated osteoblasts. These data of immunohistochemistry and micro-CT from OA model mice also demonstrated the weak staining of MMPs in cartilage and prevention of aberrant subchondral bone formation after ACY-1215 injection. Therefore, high expression of HDAC6 in osteoblasts also contributed to the OA progression, and our study provided a new evidence that HDAC6 inhibitor may be a potential therapeutic drug for OA.