TNF-α is released in a variety of pathological states in the inner ear. Inducible NO synthase (iNOS) can be induced by cytokines and other inflammatory factors, and is generally thought to be associated with inflammation and other pathological processes in the cochlea. The purpose of the present study is to reveal that TNF-α could induce apoptosis in auditory cell line and to investigate the role of nitric oxide (NO) in TNF-α-induced auditory cell death. UB-OC1 cells and zebrafish were exposed to TNF-α. Flow cytometry, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay, assay of mitochondrial membrane potential (MMP) and electron microscopy were used to show that TNF-α could induce apoptosis. Western blot was used to measure iNOS expression and Mitogen-activated protein kinase (MAPK) pathway. Flow cytometic analysis, TUNEL assay, MMP and electron microscopy all demonstrated that TNF-α could induce apoptosis in UB-OC1 cells. TNF-α significantly increased NO generation and iNOS expression. Pretreatment with iNOS blocker NG-methyl-L-arginine (NMA) attenuated TNF-α-induced cell death and caspase-3 activation. Also, TNF-α treatment increased p-p38 and pretreatment of NMA reduced this increased expression of p-p38. In conclusion, TNF-α can induce apoptosis in auditory cell line, and NO production in response to TNF-α is essential for apoptosis.