JS-K, (O²-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen 1-ium-1, 2-diolate), is a novel diazeniumdiolate-based nitric oxide donor prodrug. The present study investigated the relationship between reactive oxygen species (ROS) elevation, Ca²⁺ overload and mitochondrial disruption in JS-K-induced apoptosis. JS-K could significantly inhibit cell growth and induce apoptosis in a dose-dependent manner. Meanwhile, JS-K caused the accumulation of ROS, overload of Ca²⁺, decrease of mitochondrial membrane potential, release of cytochrome c (Cyt c) from mitochondria to the cytoplasm, increase of Bax-to-Bcl-2 ratio and activation of caspase- 9/3. NAC (an antioxidant) or BAPTA (an intracellular Ca²⁺ chelator) could partially reverse the above events, while BAPTA had little effect on the levels of ROS. Furthermore, pre-treatment with Carboxy-PTIO (a NO scavenger) significantly blocked the increasing of ROS, cytosolic Ca²⁺ and reversed the loss of mitochondrial membrane potential in JS-K-induced apoptosis. Taken together, the results suggested that NO released from JS-K could significantly induce HepG2 cell apoptosis through a ROS/Ca²⁺/caspase-mediated mitochondrial pathway.