CF101 (IB-MECA) is an adenosine A3 receptor agonist that has anti-inflammatory and pain-relieving properties. Adenosine A3 receptor activation can delay the process of Osteoarthritis(OA) and prevent the occurrence of OA. However, the mechanism of CF101 on OA is still unknown. This study aimed to investigate the effect of CF101 on rats induced by anterior cruciate ligament-transection (ACLT) and rat chondrocytes induced by IL-1ß. ACLT-induced OA rats were administered CF101, and autophagy levels were measured to determine whether CF101 had an autophagy-mediated protective effect on articular cartilage. Furthermore, the mechanism by which CF101 protected articular cartilage in IL-1ß-induced chondrocytes mimicking OA was investigated. In rats treated with ACLT, CF101 was able to delay the progression of OA, as well as reduce inflammation and type II collagen degradation factors. In addition, in vitro experiments revealed that CF101 reduced type II collagen degradation factors in OA chondrocytes. In rats treated with ACLT and OA chondrocytes, CF101 enhanced autophagy and increased the ratio of AMP/ATP and AMPK protein levels while decreasing mTOR expression. Treatment of OA chondrocytes with 3-MA prior to treatment with CF101 resulted in inhibition of autophagy factor levels, as well as increased levels of inflammatory factors and type II collagen degradation compared to the CF101 group. These findings demonstrated that CF101 could protect articular cartilage against OA by enhancing the ratio of ATP/AMP and altering the AMPK/mTOR pathway to enhance autophagy and reduce inflammation. In addition, inhibition of autophagy resulted in a reduced CF101 effect.