Osteoarthritis, affecting over 8 million people in the UK, remains a debilitating condition with limited treatment options. Current therapies primarily address symptoms and can exacerbate joint damage over time. Developing disease-modifying drugs that alleviate inflammation and promote joint regeneration is crucial for long-term patient benefit. This study investigates the potential of exosome-like nano-vesicles isolated from grapefruit juice (GEVs) as a novel therapeutic approach for osteoarthritis. GEVs possess regenerative properties and present a promising avenue for clinical translation. In this study, nano-vesicles were isolated and characterized in terms of protein quantification, size, and morphology. In vitro studies demonstrated the safety and efficacy of GEVs, showing an enhancement in human chondrocyte migratory activity of over 13%. GEVs exhibited a dual mechanism of action, reducing inflammation and oxidative stress while promoting cellular regeneration. Specifically, they reduced the expression of COX2 and PTGS2, markers associated with inflammation and pain sensitization, and enhanced the expression of antioxidant genes SD2 and GPX in osteoarthritic-like chondrocytes. Additionally, GEVs downregulated the expression of ADAMTS-5 and hypertrophic COL10 while upregulating chondrogenic markers ACAN, COL2, and SOX9. This research signifies a significant advancement in osteoarthritis therapy, offering a natural, safe, and cost-effective treatment option with the potential for long-lasting benefits. Clinical translation of GEV therapy holds promise for improving patient outcomes and reducing the burden on healthcare systems.