Reactive oxygen species (ROS) and oxidative DNA damage have been implicated in the cigarette smoke-induced cytotoxicity and genotoxicity. DNA polymerase β (polβ), a key base excision repair (BER) enzyme in repairing oxidative DNA damage, may play a crucial role in fighting against the cytotoxicity and genotoxicity of cigarette smoke. In this study, we applied a novel approach to collect cigarette smoke extract (CSE) and investigated the cytotoxic and genotoxic effects of CSE by using the mouse embryo fibroblasts that express wild-type of polβ (polβ(+/+)), null of polβ (polβ(-/-)) and overexpression of polβ (polβ(oe)). Our results showed that polβ(-/-) cells treated with CSE exhibited a higher ROS level and more DNA single-strand breaks and chromosomal aberrations than that of polβ(+/+) and polβ(oe) cells. These data suggested that polβ mediated-BER may involve in repairing the CSE-induced DNA damage and protection against the cytotoxicity and genotoxicity of CSE.