Mitomycin C (MMC) has been shown to have a therapeutic effect against human pterygium fibroblasts (HPFs) by inducing apoptosis. However, there is little data about the effect of it on plasma membrane. In the present study, the cytotoxicity of MMC to HPFs including inhibiting cell growth, inducing apoptosis and bringing about membrane toxicity was investigated. It was found that MMC could significantly suppress the proliferation of HPFs in a dose-dependent manner by CCK-8 assay. Flow cytometric analysis also revealed that treatment with MMC resulted in increased percentages of apoptotic cells in a dose-dependent manner. Membrane lipid peroxidation level, lactate dehydrogenase (LDH) leakage, membrane surface topography, and membrane rigidity alterations were investigated to assess the membrane toxicity induced by MMC. Treatment with MMC at different concentrations accelerated membrane lipid peroxidation and potentiated LDH leakage, which was consistent with disturbance of membrane surface and decrease of membrane elasticity detected by atomic force microscopy. All the above changes led to the disturbed intracellular Ca(2+) homeostasis, which was an important signal triggering apoptosis. Hence, the membrane toxicity induced by MMC might play an important role in the process of apoptotic induction and the calcium channel may be one of the apoptosis mechanisms.