Osteochondral lesions of the knee (OLK) are a common cause of knee pain and associated diseases. A new bone-marrow-derived mesenchymal stem cells technique has been developed for the treatment of OLK. 30 patients with OLK underwent arthroscopic one-step procedure. The bone marrow was harvested from the patients' posterior iliac crest and arthroscopically implanted with a scaffold into the lesion site. Clinical inspection and MRI were performed. Mean International Knee Documentation Committee (IKDC) score before surgery was 29.9 ± 13.2 and 85.4 ± 4.2 at 29 ± 4.1 months (p < 0.0005), while Knee injury and Osteoarthritis Outcome Score (KOOS) before surgery was 35.1 ± 11.9 and 87.3 ± 7.3 at 29 ± 4.1 months (p < 0.0005). Control MRI and bioptic samples showed an osteochondral regeneration of the lesion site. The one-step technique appears to be a good and reliable option for treatment of OLK at three years of follow-up. Level of evidence Case series, Level IV.