Cell therapy and tissue engineering have been intensively researched for repair of articular cartilage. In this study, we investigated the chondrogenic potential of canine adipose-derived mesenchymal stromal cells (ASCs) combined to high molecular weight hyaluronic acid (HA) in vitro, and their therapeutic effect in dogs with chronic osteoarthritis (OA) associated with bilateral hip dysplasia. Canine ASCs were characterized after conventional 2D culture or 3D culture in HA, showing adequate immunophenotype, proliferation and trilineage differentiation, as well as chondrogenesis after cultivation in HA. ASC/HA constructs were used to treat 12 dogs with OA, sequentially assigned to control, ASC and ASC/HA groups. Animals were examined for clinical, orthopedic and radiological parameters. Lameness at walk and pain on manipulation were reduced in the ASC group and mainly in the ASC/HA group. Range of motion and detection of crepitus on hip rotation and abduction improved similarly in all groups. For articular edema, muscle atrophy, Norberg angle values and radiographic analyses, there were no variations throughout the period. These results indicate that ASC/HA constructs are safe and may be an effective therapeutic tool in treating canine chronic osteoarthritis, which should be confirmed with larger studies and additional clinical parameters.