Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in not only energy metabolism and mitochondrial functions, but also calcium homeostasis and immunological functions. It has been reported that NAD(+) administration can reduce ischemic brain damage. However, the mechanisms underlying the protective effects remain unclear. Because mitochondrial impairments play a key role in the cell death in cerebral ischemia, in this study we tested our hypothesis that NAD(+) can decrease mitochondrial damage-induced cell death using differentiated PC12 cells as a cellular model. We found that NAD(+) can decrease both early-stage and late-stage apoptosis, as well as necrosis of rotenone-treated PC12 cells, as assessed by FACS-based Annexin V/AAD assay. We also found that NAD(+) treatment can restore the intracellular NAD(+) levels of the rotenone-treated cells. Moreover, NAD(+) treatment can prevent rotenone-induced mitochondria depolarization. In summary, our study has provided first direct evidence that NAD(+) treatment can prevent rotenone-induced apoptosis and necrosis. Our study has also indicated that NAD(+) treatment can prevent mitochondrial damage-induced cell death, which may at least partially result from its protective effects on rotenone-induced mitochondrial depolarization. Because both mitochondrial damage and apoptosis play key roles in multiple neurological disorders, our study has highlighted the therapeutic potential of NAD(+) for brain ischemia and other neurological diseases.