Proteostasis is critical for cell viability and as such is well-conserved throughout all organisms. In mammalian cells, newly synthesized proteins ("clients") are processed sequentially by Hsp40, Hsp70 and Hsp90 chaperones to become folded and active. Proteomic studies have uncovered a large number of post-translational modifications (PTMs) on chaperones, referred to as the "Chaperone Code". While several groups have uncovered the role and regulation of PTMs on Hsp70 and Hsp90, very little is known about the PTMs on Hsp40. In this study, we set out to clarify the role of lysine acetylation on the major yeast Hsp40, Ydj1. We mutated a series of acetylation sites on the yeast Hsp40 Ydj1 J-domain to either arginine to block any acetylation or glutamine to mimic constitutive acetylation. Cells in which acetylation was prevented appeared to have no visible phenotype, whereas acetyl-mimic mutants displayed a variety of phenotypic defects consistent with loss of Ydj1 function. Proteomic analysis of Ydj1 interactions identified 306 proteins, about 30% of which were altered in response to mutation of acetylation sites. Interestingly, proteomic data clearly showed that Ydj1 acetylation promoted dissociation with Ssa1 while only mildly altering interaction with the yeast Hsp90, Hsc82. In vitro studies confirmed that acetyl-mimic mutations on Ydj1 disrupt interaction with Ssa1 and consequently refolding of a model luciferase substrate. Excitingly, an analysis of novel Ydj1 interactors identified by MS confirmed that Ydj1 acetylation impacts the stability of key ribosomal subunits. Taken together, our results suggest that Ydj1 acetylation may be a novel regulatory mechanism to regulate translation and client maturation.