Abstract Background Cell-based therapy is being explored as an alternative treatment option for critical limb ischemia (CLI), a disease associated with high amputation and mortality rates and poor quality of life. However, therapeutic potential of uncultured adipose-derived stromal vascular fraction (SVF) cells has not been evaluated as a possible treatment. In this pilot study, we investigated the efficacy of multiple injections of autologous uncultured adipose-derived SVF cells to treat patients with CLI. Methods This study included 15 patients, from 35 to 77 years old, with rest pain and ulceration. SVF cells were injected once or twice in the ischemic limb along the arteries. Digital subtraction angiography was performed before and after cell therapy. The clinical follow up was carried out for the subsequent 12 months after the beginning of the treatment. Results Multiple intramuscular SVF cell injections caused no complications during the follow-up period. Clinical improvement occurred in 86.7% of patients. Two patients required major amputation, and the amputation sites healed completely. The rest of patients achieved a complete ulcer healing, pain relief, improved ankle-brachial pressure index and claudication walking distance, and had ameliorated their quality of life. Digital subtraction angiography performed before and after SVF cell therapy showed formation of numerous vascular collateral networks across affected arteries. Conclusion Results of this pilot study demonstrate that the multiple intramuscular SVF cell injections stimulate regeneration of injured tissue and are effective alternative to achieve therapeutic angiogenesis in CLI patients who are not eligible for conventional treatment. Trial registration number at ISRCTN registry, ISRCTN13001382. Retrospectively registered at 26/04/2017.