Chronic diabetic foot ulcers (DFUs) are a common problem in patients with diabetes and are often difficult to treat. The application of newly developed dressing material in patients with chronic DFUs has been reported to be effective. The purpose of this study was to evaluate the usefulness of allogeneic keratinocyte treatment for chronic DFUs. We performed weekly allogeneic keratinocyte treatment for up to 12 weeks in 71 patients with intractable DFUs. We investigated healing rate, wound-healing velocity, and time to 50% wound size reduction and analysed factors affecting ulcer healing. Fifty-six patients (78.8%) had complete wound healing. Forty-six patients (64.7%) showed complete healing within an average of 6.1 weeks, and 10 patients (14.1%) showed partial healing with an average 35.5% reduction vs initial size at the end of follow up. The 10 patients who showed partial healing continued to receive treatment after the 12-week study period. The mean time to complete wound healing was 7.8 weeks. Fifteen patients (21.1%) experienced treatment failure because of infection, local necrosis, no change in ulcer size, or osteomyelitis during the follow-up period. No adverse events were observed. Allogeneic keratinocyte treatment is effective for chronic, difficult-to-treat DFUs.