Mitochondrion is an organelle necessary for oxidative respiration. During industrial fermentation, brewery yeasts are exposed to long periods of hypoxia; however, the structure, role, and metabolism of mitochondria of brewery yeast during hypoxia have not been studied in detail. Our recent studies, for the first time, elucidated that the mitochondrial structure of brewery yeast can be observed throughout the brewing of sake, the Japanese traditional rice wine. As sake brewing proceeded, mitochondria of sake yeast change their morphology, which is coupled with an increase in malate production. On the basis of these insights, new fermentation technologies were developed. They include (1) breeding of low pyruvate-producing sake yeast by isolation of a mutant resistant to an inhibitor of mitochondrial pyruvate transport; and (2) modification of malate and succinate production by manipulating mitochondrial activity. These approaches provide new and practical methods to improve industrial fermentation technologies of sake brewing.