Objective To evaluate the long-term clinical and radiological outcome of matrix-assisted autologous chondrocyte implantation (mACI) for articular cartilage defects in the knee joint. Design Clinical evaluation was assessed in 21 patients with full-thickness cartilage defects, International Cartilage Repair Society (ICRS) grade IV. Clinical scoring was performed preoperatively and 12 years after transplantation using the International Knee Documentation Committee (IKDC) score, the Lysholm score, the Knee injury and Osteoarthritis Outcome Score (KOOS), and the Noyes sports activity rating scale. Morphologic evaluation of the repair tissue was assessed by magnetic resonance imaging (MRI) in 14 patients using the Kreuz-Henderson score. Results Clinical evaluation revealed significant improvement in the IKDC, the Lysholm, the KOOS, and the Noyes score. Morphological evaluation by MRI showed moderate to complete defect filling in 10 of 14 patients, demonstrating normal to nearly normal values in mean 74.29% of all assessed parameters. Significant correlation of the parameter cartilage signal and clinical outcome was found with the IKDC, Lysholm, and KOOS subscales ADL (activities of daily living) and QoL (quality of life). Conclusions The clinical and radiological outcomes 12 years after transplantation suggest the confirmation of the promising results of the mid-term follow-up. This study therefore provides first indications that the implantation of mACI might be a suitable option for long-term cartilage repair. Future controlled studies need to address the exact parameters influencing the long-term outcome of mACI.