In the past decade, mesenchymal stem cells (MSCs) have been widely used for the treatment of osteoarthritis (OA), and extracellular vesicles (EVs) may play a major role in the efficacy of this treatment. Hypoxia can change the cargo and biological functions of MSC-derived EVs (MSC-EVs). The aim of the present study was to determine whether the effects of hypoxia-preconditioned MSC-EVs on OA cartilage repair are superior to normoxia-preconditioned MSC-EVs. By using in vitro and in vivo OA models, we verified that hypoxia-preconditioned MSC-EVs improved chondrocyte proliferation and migration and suppressed chondrocyte apoptosis to a greater extent than normoxia-preconditioned MSC-EVs. Furthermore, we found that hypoxia altered the microRNA expression in MSC-EVs and identified four differentially expressed microRNAs: hsa-miR-181c-5p, hsa-miR-18a-3p, hsa-miR-376a-5p, and hsa-miR-337-5p. Bioinformatics analysis revealed that hypoxic pretreatment may promote cartilage repair by stimulating chondrocyte proliferation and migration and suppressing chondrocyte apoptosis through the miRNA-18-3P/JAK/STAT or miRNA-181c-5p/MAPK signaling pathway. Therefore, hypoxia-preconditioned EVs may be a novel treatment for OA.