Background Many treatment options for lower limb ischemia are difficult to apply for the patients with poor arterial outflow or with poor general conditions. The effect of medical treatment alone is far from ideal, especially in patients with diabetic foot. A high level amputation is inevitable in these patients. This study aimed to explore the effect of transplantation of autologous bone marrow mononuclear cells on the treatment of lower limb ischemia and to compare the effect of intra-arterial transplantation with that of intra-muscular transplantation. Methods In this clinical trial, 32 patients with lower limb ischemia were divided into two groups. Group 1 (16 patients with 18 affected limbs) received transplantation of autologous bone marrow mononuclear cells by intra-muscular injection into the affected limbs; and group 2 (16 patients with 17 affected limbs) received transplantation of autologous bone marrow mononuclear cells by intra-arterial injection into the affected limbs. Rest pain, coldness, ankle/brachial index (ABI), claudication, transcutaneous oxygen pressure (tcPO(2)) and angiography (15 limbs of 14 patients) were evaluated before and after the mononuclear cell transplantation to determine the effect of the treatment. Results Two patients died from heart failure. The improvement of rest pain was seen in 76.5% (13/17) of group 1 and 93.3% (14/15) of group 2. The improvement of coldness was 100% in both groups. The increase of ABI was 44.4% (8/18) in group 1 and 41.2% (7/17) in group 2. The value of tcPO(2) increased to 20 mmHg or more in 20 limbs. Nine of 15 limbs which underwent angiography showed rich collaterals. Limb salvage rate was 83.3% (15/18) in group 1 and 94.1% (16/17) in group 2. There was no statistically significant difference in the effectiveness of the treatment between the two groups. Conclusions Transplantation of autologous bone marrow mononuclear cells is a simple, safe and effective method for the treatment of lower limb ischemia, and the two approaches for the implantation, intra-muscular injection and intra-arterial injection, show similar results.