Abstract Menadione (MD), a simplest synthetic quinone used with reactive oxygen species (ROSs) generating agents, causes damages to Saccharomyces cerevisiae KNU5377. To understand antioxidant defense mechanisms under adaptive stress response, we have performed biochemical analyses and the genome-wide gene expression analysis after exposure to 120 μM of MD with and without adaptation. KNU5377 could survive at higher concentration of MD, 680 μM for 1 h, comparing with S. cerevisiae W303, ATCC24858 as reference strains. Glutathione peroxidase (GPX2), thioredoxin peroxidase 2 (TSA2), and thioredoxin reductase (TRR1) were up-regulated over 10-fold after treatment of 120 μM. Superoxide dismutase (SOD1), catalase (CTA1), glucose-6-phosphate dehydrogenase (ZWF1), glutathione peroxidase (GPX2, HYR1), glutathione reductase (GLR1), thioredoxin peroxidase (AHP1), and thioredoxin 2 (TRX2) were also strongly induced after adaptation. Additionally, transcriptional analysis of those genes was well corresponded with up-regulated protein expression and enzyme activity. Antioxidant defense mechanisms containing SOD1, ZWF and TRX2 maybe orchestrate by the different levels at the same time and function to produce effective defense systems. These results suggest that specific mechanisms including the three proteins were well organized for acquiring adaptive response to MD in this strain.