Intra-articular injection of platelet-rich plasma offers a promising potential for treatment of osteoarthritis in dogs. Twenty dogs weighing 25 to 50 kg (mean 38 kg) with unilateral stifle osteoarthritis were used for the study. Fourteen dogs were given intra-articular platelet rich plasma treatment and 6 dogs were used as controls. Double centrifuge method was used to obtain platelet-rich plasma. Radiography and ultrasonography of the affected joint were carried out and scores for lameness severity and pain severity were assigned by the attending clinicians. Synovial fluid was collected under sterile conditions at pre-treatment and on the 1st, 3rd, 5th, 7th, 15th days, and 4, 8 and 12 weeks after treatment. Gelatin zymography and Enzyme-linked Immunosorbent Assay were used to determine the synovial fluid levels of matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9). No adverse effects associated with the injection of the platelet concentrate or saline were observed. Hudson Visual Analog Scale and Canine Brief Pain Inventory scores for all components were non-significantly different between weeks 0, 4, 12 for control dogs. Matrix metalloproteinase-9 was totally and MMP-2 was partially inhibited in the platelet-rich plasma group. In the control group, MMP-9 was partially inhibited during the first month and activation started later. Matrix metalloproteinase-2 was constant in control samples throughout the experimental period. Platelet-rich plasma is a safe and effective method for treatment of dogs with osteoarthritis, possibly more useful for early cases with mild and moderate osteoarthritis. It is suggested that plasma rich platelet should be injected several times at regular intervals instead of a single application.