The study of signal transduction and gene expression regulation in yeast cells has contributed to understand the fundamental principle and molecular mechanism of cellular processes in higher eukaryotes. Rdr1 is a transcriptional repressor in budding yeast Saccharomyces cerevisiae, which was supposed to control pleiotropic drug resistance of cell and probably regulate genes involved in stress response. It still lacks enough evidence to conclude that Rdr1 involves in stress response. RDR1 gene was cloned by PCR and ligated into multicopy vector pYES2/NTA resulting an overexpression plasmid pYES2/NTA-RDR1. The overexpression of Rdr1 protein was induced by 2% galactose to achieve a gain-of-function phenotype. In order to reveal the function of transcription repressor Rdr1 in stress response, the growth state of wild type cells, cells with overexpressed Rdr1 protein and RDR1 mutant cells under H₂O₂, heat-shock and salt stress treatments were compared. The data demonstrated that cells with overexpressed Rdr1 protein were hypersensitive to above three stress treatments, while RDR1 mutant cells were equal or more tolerant to theses stress treatments compared to wild type cells. Yeast cells elevate antioxidant enzymes activity when challenged with different stressors. The role of RDR1 in stress response may relate to its regulation of antioxidant enzymes activity. To verify this hypothesis, the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase, glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR) were measured. It was showed that RDR1 mutant cells had higher activities of SOD, catalase, G6PDH and GR compared to wild type cells, while cells with overexpressed Rdr1 protein had lower activities of SOD, catalase, G6PDH and GR. The negative effect of RDR1 on activities of SOD and catalase was more intense than it on activities of G6PDH and GR. The results might explain the phenomenon that overexpressed Rdr1 protein were hypersensitive to above three stress treatments, while RDR1 mutant cells were equal or more tolerant to theses stress treatments compared to wild type cells. Therefore, the study provided preliminary genetics and biochemistry evidences for the negative regulatory role of transcription repressor Rdr1 in stress response.