Intra-articular injections of mesenchymal stem cells (MSCs) can inhibit the progression of osteoarthritis (OA). Previous reports have used cultured MSCs, but the ability to use thawed cryopreserved MSC stocks would be highly advantageous. Our purpose was to elucidate whether thawed cryopreserved MSCs show comparable inhibitory effects on OA progression in rats to those obtained with cultured MSCs. Cultured rat synovial MSCs or thawed MSCs were compared for in vitro viability and properties. The inhibitory effect of thawed MSCs on OA progression was evaluated by injecting cryopreservation fluid and thawed MSCs in meniscectomized rats. Cartilage degeneration was assessed using gross finding and histological scores. Cultured MSCs were then injected into one knee and thawed MSCs into the contralateral knee of the same individual to compare their effects. Cultured MSCs and MSCs thawed after cryopreservation had comparable in vitro colony formation and chondrogenic potentials. In the rat OA model, the gross finding and histological scores were significantly lower in the thawed MSC group than in the cryopreservation fluid group at 8 weeks. Finally, cartilage degeneration did not differ significantly after injection of cultured and thawed MSCs. In conclusion, thawed MSCs showed comparable inhibitory effects on OA progression to cultured MSCs.