Background: To investigate the efficacy and safety of autologous micro-fragmented adipose tissue (MF-AT) for improving joint function and cartilage repair in patients with knee osteoarthritis. Methods: From March 2019 to December 2020, 20 subjects (40 knees) between 50 and 65 years old suffering from knee osteoarthritis were enrolled in the study and administered a single injection of autologous MF-A. The data of all patients were prospectively collected. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), knee society score (KSS), hospital for special surgery (HSS) score, visual analogue score (VAS) pain score, changes in cartilage Recht grade on magnetic resonance imaging (MRI) and adverse events were analyzed before and 3, 6, 9, 12 and 18 months after injection. Results: The WOMAC, VAS, KSS and HSS scores at 3, 6, 9, 12 and 18 months after injection were improved compared with those before injection (p < 0.05). There was no significant difference in WOMAC scores between 9 and 12 months after injection (p > 0.05), but the WOMAC score 18 months after injection was worse than that at the last follow-up (p < 0.05). The VAS, KSS and HSS scores 9, 12 and 18 months after injection were worse than those at the last follow-up (p < 0.05). The Recht score improvement rate was 25%. No adverse events occurred during the follow-up. Conclusions: Autologous MF-AT improves knee function and relieves pain with no adverse events. However, the improved knee function was not sustained, with the best results occurring 9–12 months after injection and the cartilage regeneration remaining to be investigated.