Background The purpose of the present phase I clinical trial was to evaluate the safety, tolerability, and preliminary efficacy of naked DNA therapy expressing two isoforms of hepatocyte growth factor (pCK-HGF-X7) in critical limb ischemia (CLI) patients. Materials and methods Twenty-one patients with CLI were consecutively assigned to receive increasing doses (cohort I: 4mg; cohort II: 8mg; cohort III: 12 mg; and cohort IV: 16mg) of pCK-HGF-X7, which was administered into the ischemic calf and/ or thigh muscle at days 1 and 15. A safety and tolerability evaluation and measurement of pain severity score using a visual analog scale (VAS), ulcer status, transcutaneous oxygen (TcPO2) and anklebrachial index (ABI) were performed throughout a 3-month follow-up period. Results No serious adverse events were observed in any of the 21 patients for the 3-month follow-up period. A significant reduction in pain was observed in the treated patients, with the mean VAS decreasing from 5.95-1.64 (p< 0.001). The mean ABI value increased from 0.49-0.63 (p= 0.026) at 3-month follow-up. The mean TcPO2 value on the dorsum of the foot, the anterior calf and posterior calf significantly increased from 28.25-39.28mmHg (p= 0.012), from 22.00-30.63mmHg (p= 0.046) and 32.05-47.19mmHg (p= 0.001) at 3-month follow-up, respectively. Wound healing improvement was observed in the six of nine patients that had an ulcer at baseline. Conclusions These results support the performance of a phase II randomized controlled trial with pCK-HGF-X7. Copyright (C) 2011 John Wiley & Sons, Ltd.