Osteoarthritis (OA) is the most common joint disease that usually starts from joint cartilage injury. Notch2, a versatile signaling in human development and diseases, was recently uncovered to be an important regulator in chondrocyte damage. However, in OA chondrocytes, how Notch2 activation is dysregulated is largely unknown. Here, integrated bioinformatic analysis was performed on GEO datasets (GSE199193 and GSE224255) to search potential extracellular vesicles (EVs) derived regulators of Notch2 in OA chondrocytes. Ectonucleoside triphosphate diphosphohydrolase 3 (Entpd3), a most differentially expressed gene both in LPS-induced macrophage EV and Notch2 mutant chondrocytes, was screened as the candidate regulator of Notch2 in OA chondrocytes. Gain-of-function experiments in cultured human chondrocytes revealed that recombinant Entpd3 protein and macrophage EV both had a protective effect on LPS-induced inflammation, oxidative stress, apoptosis, and collagen loss in chondrocytes. In terms of mechanism, Entpd3 directly interacted with ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and suppressed AKT/Notch2-mediated mitochondrial dysfunction. Finally, we verified that either macrophage EV administration or Entpd3 overexpression was able to alleviate osteoarthritis in mice in vivo. In conclusion, Entpd3 is identified as a new regulator in OA, which alleviates mitochondrial dysfunction induced chondrocyte damage via ENPP1-induced suppression of the AKT/Notch2 pathway.