Osteoarthritis (OA) is the most common form of joint disease. The aim of this study was to explore the functions of SIRT3 on OA pathophysiology and the mechanism involved. Rat chondrocytes and destabilized medial meniscus (DMM) rat OA model were used as in vitro and in vivo models. In addition, lentivirus and plasmid were used to overexpress SIRT3, while siRNA was applied to establish SIRT3 knockdown. IL-1β induced inflammation, apoptosis, mitochondrial dysfunction, and chondrocyte degeneration were inhibited by SIRT3 overexpression, which were enhanced in SIRT3-knockdown rat chondrocytes. Furthermore, overexpression of SIRT3 could restore IL-1β-induced autophagy inhibition. We also found that IL-1β-induced PI3K/Akt/mTOR signaling pathway activation was inhibited by SIRT3 overexpression, which was enhanced by SIRT3 knockdown. Last, intra-articular SIRT3 overexpression alleviated the severity of OA-induced rat joint damage. Our results demonstrated that SIRT3 is an important protective agent against OA pathophysiology via inhibiting PI3K/Akt/mTOR signaling.