The healing of chronic lesions in patients with critical limb ischemia (CLI) is not easily achievable. Both in vitro and in vivo studies have shown that plasma mononuclear cell concentrates (MNCs), injected in the ischemic site, act through a dual mechanism: antalgic and anti-inflammatory action and neoangiogenesis stimulation. This monocentric observational clinical study evaluated the safety and efficacy of MNCs in pain reduction and wound healing in patients with CLI. From January 2017 to January 2018, there were 26 patients with CLI (grade III, category 5 Rutherford) enrolled in the study. After duplex ultrasound evaluation, 14 of 26 patients underwent a peripheral revascularization; the remaining 12 were considered not suitable for treatment. All patients underwent surgical and microbiologic examination of the ulcers. The MNCs were injected along the leg vessel’s course, in the bottom of the ulcer, and around ulcer margins. The end points, evaluated after three cycles, were healing or improvement of the lesion (according to the Texas wound classification system) and pain reduction (quantified by the numeric rating scale). In 21 patients (80%), 11 of whom revascularized, wound healing and the simultaneous disappearance of pain were achieved. In the remaining five patients, although complete ulcer healing could not be achieved, the benefit in pain control was significant. These promising preliminary results document an important antalgic, anti-inflammatory, and neoangiogenesis action of MNCs. The use of MNCs therefore represents a valid therapeutic support in patients with CLI.