Ionizing radiation (IR) induces DNA strand breaks (DSBs), base damage, inhibition of protein activity, apoptosis by reactive oxygen species (ROS). Detoxification or removal of generated ROS can reduce oxidative damage. Antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase are immediately triggered for ROS scavenging. N-acetyl-l-cysteine (NAC) having a thiol, a precursor for reduced glutathione (GSH), is known as one of the antioxidants. In this study, the effect of NAC as an antioxidant and a radioprotector was investigated on survival rate, transcriptional level of antioxidant enzymes gene, and protein level including SOD activity and intracellular GSH in yeast Saccharomyces cerevisiae W303-1A strain mutated YBP1 gene irradiated with gamma-rays. NAC did not protect the gamma-ray-induced cell death. The gene expression of antioxidant enzymes including SOD1, SOD2, GPX1, and GPX2 was induced by gamma-rays. In contrast, the pretreatment of NAC reduced the expression of these genes. NAC reduced SOD activity and intracellular GSH level in yeast. These data suggest that NAC is able to reduce radiation-induced ROS levels in vivo but does not protect yeast cells against radiation-induced death.