Effects of different pH control modes on yeast cell growth, glutathione biosynthesis and organic selenium (Se) transformation during fed-batch culture of Se-enriched Candida utilis were investigated. Based on the results derived from both flask culture and batch culture, the fed-batch culture was carried out to improve the performance of Se-enriched C. utilis. A two-stage pH control strategy of shifting pH from 3.5 to 5.5 at 12 h (pH 3.5→5.5) was found to be the optimum condition for the increase in intracellular GSH and organic Se contents of Se-enriched C. utilis. The maximal intracellular glutathione, organic selenium content and organic selenium bioconversion rate of 13.09 mg/g, 1.88 mg/g and 94.69% were achieved, respectively, under pH 3.5→5.5. According to the assay of key enzyme activity involved in glutathione biosynthesis, the measurement of intracellular oxidoreductases, and the determination of energy metabolism substance ATP, the two-stage pH control strategy of pH 3.5→5.5 was found to favor the increase in catalase activity and the decrease in malondialdehyde content of Se-enriched C. utilis. Results indicated that pH 3.5→5.5 provided appropriate intracellular redox environment for selenium assimilation and transformation, as well as glutathione biosynthesis and accumulation, all of which ultimately improved the performance of Se-enriched yeast.