Abstract: 14-3-3 proteins form a family of conserved eukaryotic proteins binding to over 200 different proteins involved in nearly all cellular processes. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. As 14-3-3 proteins are essential in most S. cerevisiae strains, we constructed a bmh mutant with suboptimal 14-3-3 protein activity. Here, we report the effect of these bmh mutations on the proteome as determined by two-dimensional gel electrophoresis and mass spectrometry. We identified 26 proteins of which the levels increased by more than 2.0-fold and 51 proteins of which the levels decreased by more than 2.0-fold in the bmh mutant compared with those of the wild-type strain. For only 9 of these proteins, a more than 2.0-fold alteration was found at the transcriptional level. The levels of many proteins involved in gluconeogenesis, including Fba1, Eno1, Eno2, Tpi1, Pck1, Mdh2, Tdh2, Tdh3, and Gpm1, increased in the mutant, whereas the levels of several proteins involved in amino acid biosynthesis and translation and heat shock proteins were lower. Our studies indicate that 14-3-3 proteins control the S. cerevisiae proteome at the post-transcriptional level, in agreement with the binding of 14-3-3 proteins to proteins involved in protein synthesis and degradation. In addition, our studies suggest a key role in the regulation of carbohydrate metabolism by 14-3-3 proteins. Keywords: 14-3-3 proteins * Saccharomyces cerevisiae * proteome * gluconeogenesis * BMH1 * BMH2.