Apoptosis mediated by caspase activation is important in the neutrophil homeostasis and resolution of tissue inflammation. Paradoxically, our previous study demonstrated that broad-spectrum caspase inhibition augmented tumor necrosis factor (TNF)-alpha-induced cell death in the human neutrophils. Therefore, we further explored the mechanisms related to the caspase-independent cell death in the neutrophils. The cell apoptosis/necrosis was determined by annexin V and propidium iodide dual staining in flow cytometry. Their morphological changes were observed under light microscopy. Fluorogenic substrates were used to measure the intracellular oxidative reactions and the activities of proteinases, calpains. Calpain inhibitors and antioxidants were used to elucidate the relationship of calpains and oxidants with the neutrophil cell death. Our results verified the caspase-independent cell death pathway in the zVAD-sensitized, TNF-alpha-stimulated neutrophils. Furthermore, the cell death was accompanied with increased calpain and oxidative activities in the cells. Calpain inhibitors, zLLY, as well as anti-oxidants, catalase and DMSO, were able to attenuate the cell death in the zVAD-sensitized, TNF-alpha-induced neutrophils. Pretreating the neutrophils with G-CSF or GM-CSF was not able to reduce the cell death. These results demonstrate that, in human neutrophils, TNF-alpha-induces a caspase-independent cell death signal, which is related to calpain and oxidative activities and cannot be rescued by the growth factor-related signaling mechanism.