The aim of the present study was to investigate the efficacy of ketamine in attenuating osteoarthritis (OA) and modulating the expression of inflammatory mediators. A rabbit OA model was established by knee immobilization using plaster bandages. After six weeks, rabbits were randomly allocated into four groups (n=6/group): Normal saline, Ket60, Ket100, and Ket200 and twice a week for four weeks the rabbits received an intra‑articular injection of saline, or 60, 100 or 200 µmol/l ketamine, respectively. One week after the final injection, samples of synovial membrane, synovial fluid and articular cartilage were isolated. The pathological changes were assessed by general observation, hematoxylin and eosin staining and Alcian blue/periodic‑acid Schiff staining. Cartilage pathology was assessed using Mankin's scoring system. Tumor necrosis factor (TNF)‑α and interleukin (IL)‑10 levels in the synovial fluid were measured by enzyme‑linked immunosorbent assays. The nuclear factor (NF)‑κB p65 subunit expression level in cartilage samples was determined by immunohistochemistry. OA was characterized by morphological changes in the articular surface, cartilage lesions, infiltration of inflammatory cells and a significantly increased Mankin's score. Elevated TNF‑α and reduced IL-10 levels in the synovial fluid, along with increased p65 expression levels in the cartilage were observed in OA rabbits. Intra‑articular injection of ketamine ameliorated the pathological characteristics of OA, reduced the Mankin's score, decreased TNF‑α and NF‑κB p65 expression levels, and increased the level of IL‑10 expression in a dose-dependent manner. Thus is was demonstrated that Ketamine suppresses the inflammatory response in OA by modulating inflammatory mediator expression levels in a rabbit model of OA.