Chondrocyte degeneration and extracellular matrix component loss are the primary causes of osteoarthritis (OA). OA can be treated by inhibiting chondrocyte degeneration and increasing extracellular matrix component secretion. Osteopontin (OPN), a multifunctional protein, has gained immense attention with regard to its involvement in OA. This study aimed to explore the therapeutic value and mechanism of action of OPN in OA treatment. Results of the histomorphological analysis revealed a worn-off OA cartilage tissue surface, cartilage matrix layer deterioration, and calcium salt deposition. Compared to that in normal chondrocytes, in OA chondrocytes, the OPN, CD44, and PI3K protein and mRNA expression was upregulated. Further, siOPN, rhOPN, and rhOPN plus LS-C179404 interfered with OA chondrocytes. As verified in mice, OPN directly inhibited the expression level of PI3K in OA chondrocytes by binding with CD44. Morphological analysis of the knee joints demonstrated that OPN effectively inhibited OA progression via the OPN/CD44/PI3K signal axis. In conclusion, OPN activates intracellular PI3K signaling molecules by binding to CD44 on the cell surface to cause downstream cascading effects, thereby delaying chondrocyte degeneration and reducing cartilage matrix component loss; therefore, OPN is a potential therapeutic agent for OA.