Objective The toxicology of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has been studied mainly with regard to the carcinogenicity of its metabolites, but its phototoxicity is not well understood. Although some studies have indicated the lethal phototoxicity of TCDD, this study was designed to investigate its effect on SPC-Al cells. Methods SPC-Al cells were cultured in 1640 medium and treated with 10 nmol/L, 0.1 μmol/L, 1 μmol/L TCDD for either 24 h or 96 h at each concentration. SPC-Al cells were co-cultured with TCDD at different concentrations. Then the cell morphology, DNA fragment electrophoresis, and cell cycle were analyzed by flow cytometry, and enzyme assays were used to observe the effect of TCDD on the morphology, growth rate, and enxyme change of SPC-A1 cells. Results With the increasing concentrations of TCDD and prolongation of culture time, the morphology of SPC-Alcells was changed from round shape to spindle, and the ability of SPC-Al cells to adhere to wall was decreased. With debris emitted around the cells, the morphologic changes included reduction in cell volume. Nuclear ehromatin condensation and PI were observed. With the increasing concentrations of TCDD, DNA ladder occurred. After treatment with TCDD, extraction of cancer cells exhibited typical DNA fragmentation, and flow eytometry analysis showed apoptosis in a dose-dependent manner. As the concentration of TCDD rose from 10 nmol/L to 1 μmol/L, the ratio of apoptotic cells increased from 10.76% to 21.82%. Conclusions TCDD has in vitro cytotoxicity on SPC-Al cells, and the cytotoxicity is positively related to its concentration and culture time. TCDD may inhibit the growth and proliferation of SPC-A lcells through the pathway of apoptosis introduction.