Background: Aryl hydrocarbon receptor (AhR) has been shown to be involved in wound healing. Objective: The aim of the study was to assess the effect of tryptophan on wound healing in vitro and in a clinical trial. Methods: The ability of tryptophan and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to increase wound healing was assessed in an in vitro scratch wound model in human keratinocytes. Topical tryptophan and vehicle were assessed for 12 weeks in 51 patients with lower limb ulcers that were resistant to conventional therapies. Results: TCDD 0.1 nM and tryptophan 10 µM increased the rate of scratch recovery in a culture model. Topical tryptophan induced a stronger pain relief and a faster reepithelialization than its vehicle in patients with lower limb ulcers. Conclusion: Tryptophan shows promising potential as a novel topical treatment for wound healing.