Osteoarthritis (OA), the most prevalent form of arthritis, affects 500 million people worldwide and is characterized by an irreversible loss of glycosaminoglycans (GAGs) at articular surfaces. Despite advances, preserving cartilage GAGs and controlling their turnover in living cells remain challenging. Based on the hypothesis that GAGs can interact with cationic molecules, we demonstrated a cost-effective strategy to increase human cartilage GAGs using a molecular machine hexadimethrine bromide (HDMBr). HDMBr promoted stem cell chondrogenesis by attracting pericellular GAGs and upregulating vesicle formation, leading to increased matrix secretion. Particularly, HDMBr promoted the assembly of chondroitin sulfate (CS) into highly concentrated condensates during intracellular trafficking, resulting in more efficient GAG secretion. HDMBr was then evaluated as a potential therapeutic in two animal models. In a rabbit model of large cartilage defects, HDMBr promoted the intrinsic regeneration of GAG-rich hyaline-like cartilage and improved tissue integration. In a rat OA model, low-dose HDMBr treatment increased cartilage thickness, supported cartilage matrix homeostasis, and improved the efficiency of cell-based therapy, evidently slowing OA progression compared to other tested clinical treatments. Overall, this study introduces a cost-effective GAG manipulation approach to cartilage repair and joint preservation, offering new insights into the mechanisms of cell-material interactions.