Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease caused by excessive external loading. Recently, it was reported that the damage in the mineralized subchondral bone caused by traumatic impact-loading is responsible for the initiation and progression of cartilage degeneration. Thus far, we have hypothesized that cytokines released from damaged subchondral bone from impact-loading affect the cartilage catabolism under pathological conditions. An impactor of 200 gw was dropped onto the top of a porcine mandibular condyle. After organ culture for 2 days, we investigated the association between the subchondral bone and cartilage using histological and biochemical experiments. The impact-loading induced the expression of IL-1β immunohistochemically and prominently up-regulated IL-1α and IL-1β mRNA levels in subchondral bone. We confirmed a significant decrease in type II collagen and aggrecan mRNA expressions in chondrocytes by co-culture with osteoblasts after impact-loading, and significant increase in mRNA and protein expressions of IL-1β in subchondral osteoblasts from impact-loaded subchondral bone. The mRNA expressions of type II collagen, aggrecan, and type X collagen in chondrocytes were decreased significantly by the co-culture with osteoblasts pre-treated by IL-1β, -6, and TNF-α. Among them, osteoblasts pre-treated by IL-1β affected chondrocytes most strongly. It was also shown that IL-1β-treated osteoblasts enhanced the MMP-1 mRNA level most markedly in chondrocytes among the four cytokines. These results suggest that the TMJ subjected to impact-loading can increase directly IL-1β synthesis in the subchondral region, subsequently altering the metabolism of adjacent cartilage and may eventually resulting in the onset and progression of TMJ-OA.