To investigate the cytotoxic effect of lidocaine on human corneal epithelial (HCEP) cells and its underlying mechanism, in vitro cultured HCEP cells were treated with lidocaine at different doses and examined by light microscopy, MTT assay, acridine orange／ethidium bromide (AO／EB) doublefluorescent staining, DNA electrophoresis, TUNEL assay, flow cytometry, and transmission electron microscopy (TEM). Results of light microscopy and MTT assay showed that lidocaine at doses of 125～1000g／L exhibited significant cytotoxicity to HCEP cells, in a doseand timedependent manner. Results of AO／EB doublefluorescent staining showed that lidocaine at doses of 0625～10000g／L elevated the plasma membrane permeability of HCEP cells, and the apoptotic rate of lidocaine-induced HCEP cells was also in a dose-and time-dependent manner. Results of DNA electrophoresis and TUNEL assay showed that lidocaine induced DNA fragmentation of HCEP cells. TEM observation showed that lidocaine induced ultrastructural changes of HCEP cells which were similar to that of apoptotic cells, such as cytoplasmic vacuolation, chromatin condensation, disordered cristae in swollen mitochondria, presence of apoptotic bodies, etc. Flow cytometry of annexin V／PI staining showed that lidocaine induced translocation of phospholipid phosphatidylserine (PS) in plasma membranes of HCEP cells. ELISA assay showed that lidocaine induced over-expression of caspase 3,8,9,10 in HCEP cells, indicating that lidocaine could induce apoptosis of HCEP cells, not necrosis. In conclusion, lidocaine at dose above 0.625g／L has significant cytotoxicity to HCEP cells in dose-and time-dependent manners, which is realized by inducing apoptosis in these cells, suggesting the inescapable sever cytotoxic side effect of lidocaine in eye clinic which should be utilized with attention.