Objective To investigate the role of osteocytic connexin43 (Cx43)-formed channels after being submitted to mechanical overloading on knee joint. Methods The strain was applied to left knee joint of 15-week Cx43 transgenic mice (R76W: gap junctions were blocked and hemichannels were promoted; Δ130-136: both hemichannels and gap junctions were blocked) and their wild-type littermates, and right knee joint was as control. A week later, the mice gait was analyzed to evaluate the structure of subchondral bone and articular cartilage.After inhibition of Cx43 channels, MLO-Y4 cells were submitted to 15% cyclic tensile strain, and the expression of related factors were detected. The supernatant was collected to culture ATDC5 cells, and the gene expression and the osteogenic differentiation ability were detected in ATDC5 cells. Results Gait analysis showed that inhibition of Cx43 channels did not significantly affect the mice gait. However, the femoral OARSI score increased significantly and the ratio of trabecular bone area decreased in transgenic mice. Conditional medium from MLO-Y4 cells with inhibition of Cx43 channels reduced the expression of ADAMTS5 and COL2 in ATDC5 cells, and enhanced the osteogenic differentiation ability. Conclusions The osteocytic Cx43 channels can respond to mechanical overloading, affect the expression of key genes and is involved in osteogenic differentiation process in chondrocytes, thus affects cartilage structure in response to mechanical overloading. The study will provide new ideas for the prevention and treatment of osteoarthritis.