The osteochondral interface is a thin layer that connects hyaline cartilage to subchondral bone. Subcellular elemental distribution can be visualised using synchrotron X-ray fluorescence microscopy (SR-XFM) (1 μm). This study aims to determine the relationship between elemental distribution and osteoarthritis (OA) progression based on disease severity. Using modified Mankin scores, we collected tibia plates from 9 knee OA patients who underwent knee replacement surgery and graded them as intact cartilage (non-OA) or degraded cartilage (OA). We used a tape-assisted system with a silicon nitride sandwich structure to collect fresh-frozen osteochondral sections, and changes in the osteochondral unit were defined using quantified SR-XFM elemental mapping at the Australian synchrotron's XFM beamline. Non-OA osteochondral samples were found to have significantly different zinc (Zn) and calcium (Ca) compositions than OA samples. The tidemark separating noncalcified and calcified cartilage was rich in zinc. Zn levels in OA samples were lower than in non-OA samples (P = 0.0072). In OA samples, the tidemark had less Ca than the calcified cartilage zone and subchondral bone plate (P < 0.0001). The Zn–strontium (Sr) colocalisation index was higher in OA samples than in non-OA samples. The lead, potassium, phosphate, sulphur, and chloride distributions were not significantly different (P > 0.05). In conclusion, SR-XFM analysis revealed spatial elemental distribution at the subcellular level during OA development.