Intracellular fluorescence lifetime and intensity images of the endogenous fluorophore of nicotinamide adenine dinucleotide (NADH) have been observed before and after application of nanosecond pulsed electric field (nsPEF) in normal and cancer cells, that is, in Wistar-King-Aptekman rat fetus fibroblast (WFB) cells and W31 cells, which are the malignant transformed cells from WFB. The application of nsPEF induces a change both in intensity and lifetime of NADH, indicating that the intracellular function is affected by application of nsPEF in both normal and cancer cells. The application of nsPEF induces an increase in the fluorescence lifetime of NADH and a morphological change, which is attributed to the induction of apoptosis by nsPEF. The field effect on the intensity and lifetime clearly depends on the pulse width, and magnitude of the field-induced increase in the fluorescence lifetime of NADH has a tendency to increase with a decreasing pulse width. It is also found that apoptosis can be induced only in cancer cells using a suitable nsPEF, showing a possibility that ultrashort pulsed electric field is applicable for drug-free cancer therapy.