Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the effect of antiepileptic lamotrigine against the cytotoxicity of mitochondrial respiratory complex I inhibitors rotenone and 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process and oxidative stress. Both rotenone and MPP+ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Lamotrigine significantly attenuated the rotenone- or MPP+-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. The preventive effect of lamotrigine against the toxicity of rotenone was greater than its effect on that of MPP+. The results show that lamotrigine seems to reduce the cytotoxicity of rotenone and MPP+ by suppressing the mitochondrial permeability transition formation, leading to cytochrome c release and subsequent activation of caspase-3. The preventive effect may be ascribed to its inhibitory action on the formation of reactive oxygen species and depletion of GSH. Lamotrigine seems to exert a protective effect against the neuronal cell injury due to the mitochondrial respiratory complex I inhibition.