AbstractArticular cartilage repair and regeneration is an unmet clinical need because of the poor self-regeneration capacity of the tissue. In this study, we found that the expression of prostaglandin E receptor 4 (PTGER4 or EP4) was largely increased in the injured articular cartilage in both humans and mice. In microfracture (MF) surgery-induced cartilage defect (CD) and destabilization of the medial meniscus (DMM) surgery-induced CD mouse models, cartilage-specific deletion of EP4 remarkably promoted tissue regeneration by enhancing chondrogenesis and cartilage anabolism, and suppressing cartilage catabolism and hypertrophy. Importantly, knocking out EP4 in cartilage enhanced stable mature articular cartilage formation instead of fibrocartilage, and reduced joint pain. In addition, we identified a novel selective EP4 antagonist HL-43 for promoting chondrocyte differentiation and anabolism with low toxicity and desirable bioavailability. HL-43 enhanced cartilage anabolism, suppressed catabolism, prevented fibrocartilage formation, and reduced joint pain in multiple pre-clinical animal models including the MF surgery-induced CD rat model, the DMM surgery-induced CD mouse model, and an aging-induced CD mouse model. Furthermore, HL-43 promoted chondrocyte differentiation and extracellular matrix (ECM) generation, and inhibited matrix degradation in human articular cartilage explants. At the molecular level, we found that HL-43/EP4 regulated cartilage anabolism through the cAMP/PKA/CREB/Sox9 signaling. Together, our findings demonstrate that EP4 can act as a promising therapeutic target for cartilage regeneration and the novel EP4 antagonist HL-43 has the clinical potential to be used for cartilage repair and regeneration.