AIM: To examine the effect of oxybuprocaine hydrochloride (OBPC-HCl) on in vitrocultured human corneal endothelial (HCE) cells and provide foundations for clinical safety use of OBPC-HCl.METHODS: After treated with OBPC-HCl at different concentrations, the growth situation and morphology of in vitrocultured HCE cells were checked under an inverted light microscope, membrane permeability was detected by acridine orange/ethidium bromide (AO/EB) double-fluorescent staining. DNA fragmentations and ultrastructure of HCE cells were observed by DNA agarose gel electrophoresis and transmission electron microscopy (TEM), respectively. RESULTS: In vitrocultured HCE cells treated with OBPC-HCl at a concentration between 62.5mg/L and 4g/L showed typical characteristics of apoptosis, including cell shrinkage, intercellular vacuolation, membrane permeability increasing, chromatin condensation, apoptotic body appearance and DNA fragmentation. The apoptosis-inducing effect of OBPC-HCl was in dose- and time-dependent manners. The greatest apoptosis-inducing effect of OBPC-HCl on HCE cells was found at the clinic concentration of 4g/L, and the apoptotic rate of HCE cells reached 100% after treated for 1 hour. CONCLUSION: OBPC-HCl at a concentration of 62.5mg/L-4g/L has an obvious apoptosis-inducing effect on HCE cells, and has huge toxic side effects on HCE cells during its clinic ophthalmic usage.