4-Methoxy-TEMPO, a derivative of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), is a stable nitroxide radical and is generally used in organic and pharmaceutical syntheses for the oxidation of alcohols. Previously, we reported the involvement of reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) in TEMPO-induced apoptosis in mouse L5178Y cells. In this study, we investigated 4-methoxy-TEMPO induced toxicity in human HepG2 hepatoma cells and its underlying mechanisms. Treatments with 4-methoxy-TEMPO (0.5-5 mM for 2-6 h) caused oxidative stress as demonstrated by increased intensity of the ROS indicator HDCF-DA, decreased levels of glutathione. 4-Methoxy-TEMPO treatment also induced DNA damage as characterized by increased levels of DNA tail intensity in the Comet assay, increased phosphorylation of related proteins including γ-H2A.X, p-Chk1, and p-Chk2, and activation of MAPK signaling pathways. In addition, 4-methoxy-TEMPO also induced autophagy as demonstrated by the conversion of LC3B-I to II, decreased level of p62, and the appearance of GFP-LC3B punctae. To investigate the crosstalk between different signaling pathways, pretreatment of HepG2 with N-acetylcysteine, an ROS scavenger, attenuated 4-methoxy-TEMPO-induced DNA damage, suppressed JNK activation, and diminished autophagy induction. Furthermore, inhibiting JNK activation by a JNK-specific inhibitor, SP600125, decreased DNA damage levels induced by 4-methoxy-TEMPO. These results suggest that multiple mechanisms including ROS generation, DNA damage, and MAPK activation contribute to 4-methoxy-TEMPO-induced toxicity.