Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein– protein interactions, a global view of theHsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins onHsp90,we used the \"stable isotope labeling by amino acids in cell culture\"method coupled withmass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells.We observed that 904 proteins changed in their abundance by more than 1.5-fold.When compared with the transcriptome of the same population of cells, two-thirds of themisregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networkswith a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes.