Objective The aim of this study was to investigate the safety, feasibility and clinical efficacy of a single intra-articular injection of autologous and purified micro-fragmented adipose tissue for the treatment of osteoarthritis (OA) in dogs. Study Design Twenty-one client-owned dogs with radiographically confirmed OA were recruited into this prospective study. Lameness and discomfort were evaluated by physical examination at day 0 and then 14, 30, 60 and 180 days after injection. Kinetic data and temporospatial parameters were obtained using a pressure-sensing walkway. Peak vertical force, vertical impulse and percentages of body weight distribution were determined. Owner perception data regarding their own dog's physical activity were also collected using the Canine Brief Pain Inventory. Results Radiographic scores for OA from days 0 to 180 were similar, except in two dogs. No major side effects were noted after injection. Lameness and Canine Brief Pain Inventory scores were significantly lower at all time points compared with day 0. Post-injection results demonstrated gradual improvement of kinetic data up to day 180 compared with pre-treatment values: vertical impulse (>2.25%), peak vertical force (>5.32%) and percentages of body weight distribution (>3.6%). In dogs with elbow OA, gait analysis values significantly increased at all time points compared with day 0. Conclusion Regenerative autologous adipose tissue injection therapy is a promising alternative to traditional analgesics treatment in patients with OA, associated with significant reductions in pain and lameness, delayed disease progression and improved quality of life.