The effects of a fractionated 70% ethanolic extract of propolis were analyzed at the subproteome level by two-dimensional electrophoresis. Differential detergent fractionation was used to fractionate proteins from the yeast Saccharomyces cerevisiae according to their subcellular localization. Thus, four subcellular proteomes were obtained: cytosolic, membrane/organelle, nuclear, and cytoskeletal. Yeast treatment resulted in changes in the levels of proteins involved in carbohydrate and energy metabolism, antioxidant defense, actin filament dynamics, folding of proteins, and others. On the basis of this information, we can obtain better insights into the processes that are carried out in cells exposed to propolis extract.