Aims: A major problem in industrial fermentation of organic acids with micro-organisms is to ensure a suitable pH in the culture broth. To circumvent this problem, we investigated the effect of citrate, which is a widely used auxiliary energy co-substrate, on cell growth, organic acid production and pH homeostasis among extracellular environment, cytoplasm and vacuole, in the pyruvic acid production by Candida glabrata CCTCC M202019 under different pH conditions. Methods and Results: Analysis of intracellular ATP regeneration, cytoplasmic and vacuolar pH values under different culture conditions points towards a relief of stress when C. glabrata is exposed to lower pH, if citrate is added. When 50 mmol l-1 citrate was added to the culture medium, the intracellular ATP concentrations increased by 20 center dot 5% (pH 5 center dot 5), 20 center dot 4% (pH 5 center dot 0) and 39 center dot 3% (pH 4 center dot 5), and higher pH gradients among the culture broth, cell cytoplasm and vacuoles resulted. As a consequence, the cell growth and pyruvic acid production of C. glabrata CCTCC M202019 were significantly improved under pH 5 center dot 0 and 4 center dot 5. Conclusions: The acid tolerance of yeast can be improved by enhancing the ATP supply, which helps to maintain higher pH gradients in the system. Significance and Impact of the Study: The results presented here expand our understanding of the physiological characteristics in eukaryotic micro-organisms under low pH conditions and provide a potential route for the further improvement of organic acids production process by process optimization or metabolic engineering.