Olaquindox, a synthetic antimicrobial compound, is widely used in China as feed additive for growth promotion. However, it is a mutagen with its functional mechanism yet to be unclear. The purpose of this study was to investigate the genotoxic effects of olaquindox in human hepatoma G2 (HepG2) cells and to determine whether the oxidative DNA damage participated in the mechanism of olaquindox toxicity. The results of cell survival assay revealed that the HepG2 cells viabilities were significantly inhibited by olaquindox in a dose- and time-dependent manner. The cytokinesis-block micronucleus (CBMN) assay demonstrated a clear dose-response relationship between olaquindox treatments and micronucleus (MN) frequencies. Moreover, marked increases of DNA fragment migration were observed in the single cell gel electrophoresis (SCGE) assay. These data suggest that olaquindox treatment produced serious chromosome damage and DNA damage in HepG2 cells. To elucidate the possible oxidative DNA damage mechanism of olaquindox genotoxic activity, the levels of the intracellular reactive oxygen species (ROS) and the formation of 8-hydroxydeoxyguanosine (8-OHdG) were detected. The results showed that olaquindox induced the increased levels of ROS and 8-OHdG in HepG2 cells. Considering all the results, it is inferred that olaquindox exerts genotoxic effects in HepG2 cells probably through the ROS-induced oxidative DNA damage.