Several cell lines can be used in the in vitro micronucleus (MN) assay to evaluate the genotoxic potential of various chemicals. However, less information is available on cell types suitable for genotoxic assessment of cigarette smoke. Therefore, we compared the potential of MN induction by cigarette smoke in L5178Y, TK6, and CHL/IU cell lines. The total particulate matter of reference cigarette (3R4F) smoke was suspended in DMSO as a test sample. After 3 h of treatment followed by 24 h of recovery, a dose-dependent MN increase was observed in all cell lines. However, the lowest concentrations with significant genotoxicity and cytotoxicity differed among the cell lines. 3R4F smoke generates reactive oxygen species (ROS). Therefore, we explored the relationship between the different sensitivities to 3R4F smoke in each cell line and their antioxidant capacity. Total antioxidant capacity and cellular glutathione (GSH) were higher in CHL/IU cells than in L5178Y cells. Furthermore, pre-treatment of CHL/IU cells with N-acetylcysteine (NAC) or buthionine sulfoximine (BSO), a precursor and inhibitor of GSH biosynthesis, respectively, resulted in higher or lower 3R4F concentrations for significant genotoxicity and cytotoxicity. The same effect exerted by NAC or BSO was also seen after treatment with allyl isothiocyanate, a ROS-generating chemical, whereas treatment with mitomycin C, a genotoxin without ROS generation, was not affected by NAC or BSO pre-treatment. Pre-treatment with NAC increased the cellular thiol level, which may have prevented genotoxicity and cytotoxicity caused by 3R4F or allyl isothiocyanate. These results indicate that the degrees of genotoxicity and cytotoxicity induced by cigarette smoke differ among these cell lines and such differences might be related to the antioxidant capacity of the employed cell line.