AIM: To evaluate the cytotoxicity of a novel anticholinergic drug penehyclidine hydrochloride (PHC) and its four optical isomers R-1, R-2, S-1, and S-2. METHODS: Two in vitro assays, MTT assay and neutral red uptake assay, were used to evaluate the cytotoxicity following PHC and its isomers exposure to HepG2 cells at different concentrations. RESULTS: PHC and its isomers induced decreases of viability of HepG2 cells in a concentration-dependent manner. Comparison of the cytotoxicity of the five anticholinergic agents with 50% inhibitory concentration (IC50) values indicated that the order of potency was PHC>R-2>R-1>S-2>S-1 for MTT assay, and R-2>PHC≈R-1>S-2>S-1 for neutral red uptake assay. CONCLUSION: With respect to the cytotoxicity of the four isomers on HepG2 cells, the R configuration was more potent than the S configuration, and R-2 was the most potent isomer whereas S-1 was the least potent isomer among the four optical isomers.