AbstractOsteoarthritis (OA) is characterised by the deterioration of cartilage in the joints and pain. We hypothesise that semaphorin‐3A (sema‐3A), a chemorepellent for sensory nerves, plays a role in joint degradation and pain. We used the mechanical joint loading (MJL) model of OA to investigate sema‐3A expression in the joint and examine its association with the development of OA and pain. We also analyse its effect on chondrocyte differentiation using the ATDC5 cell line. We demonstrate that sema‐3A is present in most tissues in the healthy joint and its expression increases in highly innervated tissues, such as cruciate ligaments, synovial lining and subchondral bone, in loaded compared to nonloaded control joints. In contrast, sema‐3A expression in cartilage was decreased in the severe OA induced by the application of high loads. There was a significant increase in circulating sema‐3A, 6 weeks after MJL compared to the nonloaded mice. mRNA for sema‐3A and its receptor Plexin A1 were upregulated in the dorsal root ganglia of mice submitted to MJL. These increases were supressed by zoledronate, an inhibitor of bone pain. Sema‐3A was expressed at all stages of Chondrocyte maturation and, when added exogenously, stimulated expression of markers of chondrocyte differentiation. This indicates that sema‐3A could affect joint tissues distinctively during the development of OA. In highly innervated joint tissues, sema‐3A could control innervation and/or induce pain‐associated neuronal changes. In cartilage, sema‐3A could favour its degeneration by modifying chondrocyte differentiation.