The present study was designed to evaluate the safety and potential of adipose tissue-derived stem cells (ASCs) for the treatment of Crohn's fistula. In this dose escalation study, patients were sequentially enrolled into three dosing groups with at least three patients per group. The first three patients (group 1) were given 1 × 10(7) cells/ml. After 4 weeks, this dose was deemed safe, and so an additional four patients (group 2) were given 2 × 10(7) cells/ml. Four weeks later, after which this second dose was deemed safe, a third and final group of three patients were given 4 × 10(7) cells/ml. Each patient was followed for a minimum of 8 weeks. Patients who showed complete healing at week 8 were followed up for an additional 6 months. Efficacy endpoint was complete healing at week 8 after injection, defined as complete closure of the fistula track and internal and external openings without drainage or signs of inflammation. There were no grade 3 or 4 severity adverse events, and there were no adverse events related to the study drug. Two patients in group 2, treated with 2 × 10(7) ASCs/ml, showed complete healing at week 8 after injection. Of the three patients enrolled in group 3, treated with 4 × 10(7) ASCs/ml, one showed complete healing. Outcome in another patient was assessed as partial healing due to incomplete closure of the external opening, although the inside of fistula track was filled considerably and there was no drainage. All three patients with complete healing at week 8 showed a sustained effect without recurrence 8 months after injection. In conclusion, this study demonstrates the tolerability, safety, and potential efficacy of ASCs for the treatment of Crohn's fistula and provides support for further clinical study.