Background: There is limited study about the human umbilical cord-blood derived mesenchymal stem cell (hUCB-MSC) cartilage regeneration procedures combined with high tibial osteotomy (HTO). We compared the clinical and radiological results of hUCB-MSC cartilage regeneration procedures combined with HTO to those of microfracture with HTO. Methods: From August 2017 to December 2018, HTO patients with International Cartilage Regeneration and Joint Preservation Society (ICRS) grade IV cartilage defects over 200 mm 2 on medial femoral condyle (MFC) were enrolled. For comparison, all participants were divided into two groups: those who had undergone an hUCB-MSC induced cartilage regeneration procedure (group MSC) and those with microfractures only (group C, controls). Clinically, Hospital for Special Surgery (HSS), International Knee Documentation Committee (IKDC), and Lysholm scores were evaluated post-operatively at 18 months. Radiologically, mechanical axis (MA) and joint space width (JSW) were evaluated. Results: A total of 100 knees were enrolled (43 in group MSC, 57 in group C). The IKDC score in group MSC (69) was better than that in group C (62; P < 0.05). The JSW increment in the MSC group (0.6 mm) was more than that in group C (0.1 mm; P < 0.05). No patient developed nonunion, correction loss, or arthroplasty conversion. Conclusion: hUCB-MSCs can improve clinical outcome and JSW better than microfracture only in HTO patients.