Peroxisomes are organelles that are crucial for cellular metabolism, but they also play important roles in non-metabolic processes such as signalling, stress response or antiviral defense. To uncover the consequences of peroxisome deficiency, we compared Saccharomyces cerevisiae wild-type with pex3 cells, which lack peroxisomes, employing quantitative proteomics and transcriptomics technologies. Cells were grown on acetate, a carbon source that requires peroxisomal enzymes of the glyoxylate cycle to generate energy and essential carbohydrates, and that does not repress the expression of peroxisomal genes. Our integrative omics analysis reveals that the absence of peroxisomes induces distinct responses at the level of the transcriptome and proteome. Transcripts of genes and corresponding proteins that are associated with peroxisomal β-oxidation were mostly increased in pex3 cells. In contrast, levels of peroxins were regulated at protein but not at transcript level. Membrane-bound peroxins were reduced, whereas the soluble receptors Pex5 and Pex7 were increased in abundance in pex3 cells. Interestingly, we found several non-peroxisomal transcript and proteins regulated in pex3 cells including mitochondrial proteins involved in respiration or import processes, which led to the identification of the mitochondrial pyruvate carrier Mpc1/3 as so far unnoticed transporter present in the peroxisomal membrane. Our results reveal the impact of the absence of peroxisomes in pex3 yeast cells and represent a rich resource of genes/proteins for follow-up studies to obtain a deeper understanding of peroxisome biology in a cellular context.