Calcium ion (Ca2+), which serves as one of the important signaling agents, is imperative to control normal cell function. The altered Ca2+ signaling is known to play a crucial role in the resistance of cancerous cells against apoptosis. Herein, nanosecond pulsed electric field (nsPEF) was applied to non-small lung cancerous cells A549, and Ca2+ mobilization along with mitochondria-mediated apoptotic cell death that accompanies morphological changes was investigated. In the presence of nsPEF, intracellular mobilization of Ca2+ ions through the efflux from endoplasmic reticulum storage was dependent on the pulse-width of the applied field. The field-induced Ca2+ mobilization has shown to correlate with the superoxide anion (O2−) generation, which probably occurred in mitochondria utilizing the nicotinamide adenine dinucleotide (NADH), as supported by the field-induced change in autofluorescence intensity and lifetime of NADH. Mitochondrial dysfunction and O2− generation, which promoted apoptotic cell death, were also induced by nsPEF with strong correlation with intracellular Ca2+ ions.