We investigated morphological changes of mitochondria and the actin cytoskeleton in the yeast Saccharomyces cerevisiae during diauxic growth and glucose depletion culture. S. cerevisiae exhibits diauxic growth during aerobic culture in a medium containing glucose as a carbon source. We demonstrated that mitochondria in budded cells maintain a tubular morphology at the log phase, but begin to fragment at the diauxic shift phase, in which the metabolism in cells switches from fermentation to respiration due to glucose depletion. On the other hand, the actin cytoskeleton exhibited a polarized distribution in budded cells at the log phase but rapidly depolarized at the diauxic shift phase. The fragmentation of mitochondria and depolarization of the actin cytoskeleton also occurred when cells at the log phase were transferred into a glucose-free medium. In these studies, we found that rapid Dnm1p-dependent fragmentation of mitochondria occurs via glucose depletion. In addition, the results suggest that the changes in mitochondrial morphology and in the polarity of the actin cytoskeleton are independent of each other.