To evaluate the long-term safety and efficacy of adipose-derived mesenchymal stem cell (ADMSC) therapy in the treatment of knee osteoarthritis (OA). 329 participants with knee OA underwent intra-articular ADMSC therapy. Participants were followed up for 24 months and were separated based on radiological OA grade. Treatment was well tolerated with no related serious adverse events. All participant groups reported clinically and statistically significant pain improvement. Clinical outcome was not influenced by patients' age or BMI. ADMSC therapy is an effective, safe and long-lasting treatment option for knee OA with the potential to delay total joint replacement. In addition to the observed clinical benefits, ADMSC therapy promises to reduce the global economic burden of OA. Trial registration number: ACTRN12617000638336.