Osteoarthritis (OA) is one of the most common degenerative diseases in dogs and humans, which can lead to articular cartilage deterioration, chronic pain, and decreased quality of life. The anti-inflammatory, anti-fibrotic, analgesic, and cartilage regeneration properties of mesenchymal stem cell (MSC) therapy provide a new direction for the treatment development of OA in the future. Currently, MSC therapy lacks confirmed ideal sources, dosages, formulations, and specific characteristics. In this study, we evaluated the efficacy of multiple canine adipose-derived mesenchymal stem cell (ADSC) injections on anti-inflammation and joint cartilage damage in a canine OA model. Considering animal ethics, we simulated the effects of inflammation and cartilage repair during treatment through a mouse OA model. In the mouse OA model, through the detection of cartilage repair and inflammation-related key factors via histology and molecular biology, it was found that MSC therapy has a certain repair effect on cartilage, but the anti-inflammatory effect is time-dependent. In the canine OA model, we verified the feasibility of multiple injections of ADSCs. Compared with the control group, the cartilage repair effect of the treatment group was obvious, and the inflammatory factors decreased, showing an obvious therapeutic effect. This study demonstrates that multiple intra-articular injections of canine ADSCs could be effective in treating OA symptoms.