The osteochondral interface is a thin layer in adults connecting hyaline cartilage and subchondral bone. The osteochondral interface undergoes significant changes during osteoarthritis (OA) progression. Previous studies mainly focus on cartilage and subchondral bone but underestimated the function and changes of osteochondral interface. The morphological, molecular, and biochemical changes of the osteochondral interface have not been fully understood yet. The aim of this study is to investigate the morpho-molecular changes of the osteochondral interface during OA progression. Based on the OARSI grading, the G1 and G4 knee osteochondral interface samples were selected from the medial and lateral parts of the tibial plates from 6 patients undergoing total knee replacement, and 10 medial tibia plates from rats undergoing sham or meniscectomy surgery, respectively. H&E staining, Safranin-O staining, immunohistochemistry, Scanning electronic microscopy, Energy-dispersive X-ray spectroscopy, Transmission electron microscopy, Fourier-transform infrared spectroscopy, Nanoindentation, Laser capture microdissection assisted proteomics were used to explore the characteristics of the osteochondral interface. Our results demonstrated that the osteochondral interface undergoes significant morphological and molecular changes, including the thinning of the calcified cartilage zone, loss of collagen and proteoglycan, occurrence of the endochondral ossification and neuro-vasculature, loss of the elastic module, loss of the collagen direction, increase of the tortuosity and the change of the protein expression in the region. The calcium/Phosphate ratio was not changed during the OA progression, but the calcium-binding protein and cadherin binding protein, as well as carbohydrate metabolism-related proteins, undergo significant changes during OA progression. These results suggest that the osteochondral interface undergoes significant changes driven by the changed expression of the proteins in this zone. Alleviating or reversing the pathological changes osteochondral interface could be conducive to treating OA.