Selenium may influence the biosynthesis of individual proteins in the yeast cell cytosol. In this study, we used two-dimensional (2D) electrophoresis to identify proteins that are differentially expressed by the enrich- ment of selenium in Saccharomyces cerevisiae yeast cells. We chose eight protein fractions for further proteomic analysis. A detailed analysis was performed using the Ultraflextreme matrix-assisted laser desorption/ionisa- tion time-of-flight/time-of-flight mass spectrometer, which enables fast and accurate measurement of the molecular weight of the analysed proteins. This study, for the first time, provides evidence that selenium-enriched yeast con- tains higher levels of mitochondria malate dehydrogenase, adenosine-5′-triphosphate (ATP)-dependent RNA helicase dbp3, and tryptophan dimethylallyltransferase, and alanyl- tRNA editing protein AlaX than yeast without the addition of selenium. It should be emphasised that the proteomic variability obtained reflects the high biological and com- plexity of yeast metabolism under control and selenium- enriched conditions and can be properly used in the future as a model for further research aimed at determining the expression of appropriate metabolic genes.