Despite the efforts made for osteoarthritis (OA) treatment, the results are limited and can be improved by enhancing the OA homing strategy. Here, we used a phage display system to identify OA-targeting peptides, and combined them with magnetic resonance imaging detection reagents to expand their application to early OA diagnosis in rat and swine models. OA-targeting peptides showed better static and kinetic friction characteristics than scrambled peptides, when conjugated with hyaluronic acid for rheological lubrication studies using human OA specimens. Furthermore, mesenchymal stem cells, through CD44 binding to hyaluronic acid conjugated with OA-targeting peptides, showed better capacity for OA homing and repair than those conjugated with scrambled peptides. Protein–peptide docking revealed WXPXW as the consensus binding motifs that bind to collagen XII, a protein exclusively expressed in human and animal OA models. These results suggest the potential of OA-targeting peptides to promote diagnosis, treatment, and regenerative medicine for OA.