Abstract: Objective To investigate the effect of 0.1% dimethyl sulfoxide （DMSO） on photodynamic reaction in a human keratinocyte cell line, HaCaT, induced by 5-aminolevulinic acid. Methods HaCaT cells were cultured in vitro with or without the presence of 0.1% DMSO at 37 ℃ and stimulated by δ-aminolevulinic acid （ALA） for 3 hours with light free. Then, cellular protoporphyrin Ⅸ （PpⅨ） concentration in, fluorescence intensity in, proliferation and survival rates of HaCaT cells were determined by high performance liquid chromatography with fluorescence detection （HPLC-FLD）, laser scanning confocal microscopy （LSCM）, MTT colorimetric method and trypan blue staining, respectively. Also, a portion of HaCaT cells were treated with ALA-based photodynamic therapy （ALA-PDT） and irradiated by 632.8-nm laser, and 12 hours later, cellular apoptosis and necrosis were detected by flow cytometry with annexinⅤ/PI staining. Results Increased concentrations of PpⅨ were found in DMSO-pretreated HaCaT cells compared with untreated cells （0.57 ± 0.05 μg/L vs 0.44 ± 0.04 μg/L, P < 0.05）, while no statistical difference was observed in fluorescence intensity expressed as the absorbance at 570nm （0.54 ± 0.06 vs 0.51 ± 0.07, t = 1.51, P > 0.05） or cell survival rate （（96.18 ± 2.25）% vs（94.64 ± 2.40）%, χ2 = 1.84, P > 0.05） between DMSO-pretreated and untreated cells. After ALA-PDT, the apoptosis and necrosis rate were significantly increased in DMSO-treated HaCaT cells compared with untreated HaCaT cells （2.2% vs 1.5%, χ2 = 4.05, P < 0.05; 8.9% vs 0.1%, χ2 = 8.23, P < 0.05）. Conclusion Low concentration （0.1%） of DMSO could enhance the effect of ALA-PDT on HaCaT cells.