Cisplatin is a chemotherapeutic agent that is widely used to treat cancers such as head and neck squamous cell carcinoma (HNSCC). Previously, we have reported that cisplatin induced an early caspase-dependent apoptosis (8 hr) in a HNSCC cell, HN4. In this study, we examined a late caspase-independent apoptosis as well as an early caspase-dependent apoptosis in cisplatin-treated HN4 cells. While z-VAD-fmk, a pan-caspase inhibitor, blocked the caspase activities and protected cells from the early apoptosis, it did not provide protection against delayed apoptosis occurring after extended exposure (16 hr) to cisplatin, suggesting that the delayed apoptotic response in the presence of z-VAD-fmk was caspase-independent. Cisplatin treatment induced reactive oxygen species (ROS) generation, loss of the mitochondrial membrane potential (MMP) and nuclear translocation of endonuclease G (EndoG). Small interfering RNA mediated-knockdown of EndoG significantly protected cells from the delayed apoptosis induced by cisplatin in the presence of z-VAD-fmk. Overexpression of Bcl-2 in HN4 cells prevented loss of MMP, nuclear translocation of EndoG and protected cells from the delayed apoptosis induced by cisplatin in the presence of z-VAD-fmk. Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented both ROS generation, loss of the MMP and nuclear translocation of EndoG. Together, our data indicate that cisplatin treatment induced ROS-mediated loss of the MMP, and, then, the nuclear translocation of EndoG, which played a crucial role in caspase-independent apoptosis of HN4 cells in the presence of z-VAD-fmk. This is the first report about the involvement of EndoG in cisplatin-induced caspase-independent apoptosis of cells.