Background/aims: Inflammatory mediators contribute to the impairment of vasculogenesis by reducing endothelial progenitor cells (EPCs) mobilization in atherosclerotic vasculopathy. We tested the hypothesis that administration of an oxygen/ozone mixture (IMT) might counteract this pathophysiological mechanism and enhance limb tissue perfusion in patients with critical limb ischemia (CLI). Methods: Randomized patients with rest pain or ischemic ulcers and transcutaneous oxygen tension (TcPO2) <40mmHg and/or toe pressure <50mmHg received placebo (n = 74) or a non-specific immunomodulation therapy (IMT) (n = 77), autologous blood exposed to oxygen/ozone gas mixture by intragluteal injection, on day 1, 2, 7, and once a week thereafter for at least 22 weeks. Patients were evaluated for changes in TcPO2, levels of circulating EPCs (CD34/KDR-positive cells) and inflammation (tumor necrosis factor-—TNF-). Results: TcPO2 and CD34/CD133-positive cells increased at 22 weeks in IMT group (P < 0.01) whereas no changes were observed in placebo group. TNF- levels decreased at 6 months in IMT group (P < 0.001) whereas no changes were observed in placebo group. There was a strong positive correlation between CD34/KDR-positive cells and TcPO2 (r = 0.56, P < 0.01). Moreover, therewasan inverse correlation between CD34/KDR-positive cells and TNF- (r =−0.51, P < 0.01). Conclusions: Intramuscular injection of IMT may improve wound healing and limb salvage in patients with CLI.