Glycogen is a highly branched polyglucan utilized as a carbohydrate reserve in major living systems. Industrially, it is used as a prebiotic and in the nanoencapsulation of drugs and nutraceuticals. In this study, optimal fermentation conditions enabling the highest glycogen accumulation in Saccharomyces cerevisiae were experimentally evaluated for possible mass production. Production efficiency was assessed by comparing specific growth rates, specific glycogen production rates, and glycogen yields under each condition. The results demonstrated that fermentation at 30 °C with an aeration rate of 3 vvm using a medium containing 120 g/L glucose without ethanol was optimal for robust cell growth and maximum glycogen yield. Additionally, a rich medium outperformed a minimally defined medium, and a single sugar carbon source, as opposed to mixed sugars, resulted in significantly higher cell growth and glycogen yields (p S. cerevisiae.