Abstract Background Chronic limb-threatening ischemia (CLTI) represents the final stage of peripheral arterial disease. Approximately one-third of patients with CLTI are not eligible for conventional surgical treatments. Furthermore, patients with advanced-stage of CLTI are prone to amputation and death. Thus, an effective therapeutic strategy is urgently needed. In this context, autologous bone marrow mononuclear cell (auto-BM-MNC) and allogeneic mesenchymal stem cells represent a promising therapeutic approach for treating CLTI. In this study, we compared the safety and beneficial therapeutic effect of auto-BM-MNC vs. allogeneic Wharton jelly-derived mesenchymal stem cells (allo-WJ-MSCs) in diabetic patients with CLTI. Methods We performed a randomized, prospective, double-blind and controlled pilot study. Twenty-four diabetic patients in the advanced stage of CLTI (4 or 5 in Rutherford’s classification) and a transcutaneous oxygen pressure (TcPO 2 ) below 30 mmHg were randomized to receive 15 injections of (i) auto-BM-MNC (7.197x10 6 ± 2.984 x10 6 cells/mL) ( n =7), (ii) allo-WJ-MSCs (1.333 x10 6 cells/mL) ( n =7) or (iii) placebo solution (1 mL) ( n =10), which were administered into the periadventitial layer of the arterial walls under eco-Doppler guidance. The follow-up visits were at months 1, 3, 6, and 12 to evaluate the following parameters: (i) Rutherford’s classification, (ii) TcPO 2 , (iii) percentage of wound closure, (iv) pain, (v) pain-free walking distance, (vi) revascularization and limb-survival proportion, and (vii) life quality (EQ-5D questionnaire). Results No adverse events were reported. Patients with CLTI who received auto-BM-MNC and allo-WJ-MSCs presented an improvement in Rutherford’s classification, a significant increase in TcPO 2 values, a reduction in the lesion size in a shorter time, a decrease in the pain score and an increase in the pain-free walking distance, in comparison with the placebo group. In addition, the participants treated with auto-BM-MNC and allo-WJ-MSCs kept their limbs during the follow-up period, unlike the placebo group, which had a marked increase in amputation. Conclusions Our results showed that patients with CLTI treated with auto-BM-MNC and allo-WJ-MSCs conserved 100% of their limb compared to the placebo group where 60% of participants underwent limb amputation in different times. Furthermore, we observed a faster improvement in the allo-WJ-MSC group, unlike the auto-BM-MNC group. during 12 months of the follow-up Trial registration The study was registered at ClinicalTrials.gov (NCT05631444).