Photodynamic therapy (PDT) positively influences wound healing in animal models but the mechanism is unknown. We have examined for the influence of PDT on production of TGF-β isoforms, key regulators of matrix deposition and remodelling, alongside clinical and microscopic parameters of healing, in a novel excisional wounding study in human skin. Wounds were created by 4mm punch biopsy in duplicate on the inner upper arms of healthy older men (60-77 years, n=27, phototype I-III). Following randomisation, one wound was treated immediately with methyl aminolevulinate (MAL)-PDT with repeat of this intervention on days 2 and 4, while the control wound was untreated. Wounds were re-excised by 6mm punch biopsy at 7 days (n=10 subjects), 3 weeks (n=8) and 9 months (n=9) and assessed histologically, immunohistochemically and by Western blot, for effects on the inflammatory phase, matrix deposition/remodelling phase and scarring, respectively. At 7 days, MAL-PDT resulted in delayed healing with significantly reduced re-epithelialisation (35% vs 94% in control wounds, p<0.05) and fewer cells/mm2 producing TGF-β1 (mean±SEM 1332±179 vs 2154±347 in control wounds, p<0.05). However, at 3 weeks, treated wounds appeared smaller in area (3.1±0.2 vs 3.8±0.3 cm2, p=0.07) with significantly more cells mm-2 producing TGF-β1 (975±120 vs 602±65) and TGF-β3 (1677±250 vs 862±163; both p<0.05), without change in TGF-β2 production. Relative production of TGF-β3 protein was also higher (p<0.01). Treated wounds were significantly closer in clinical appearance to normal skin (p=0.05). At 9 months, MAL-PDT treated wounds showed less collagen I and III deposition (both p<0.05), a matrix deposition pattern closer in resemblance to that of normal skin, and a trend to improved cosmesis. Thus, MAL-PDT positively influences wound healing in human skin, with upregulation of TGF-β1 and 3 during the matrix deposition and remodelling phase providing a mechanism for the observed improved healing and more ordered collagen structure of scars.