In order to investigate rapid non-genomic effects of acute stress, rats were restrained for 15 min which was sufficient to activate the hypothalamus-pituitary-adrenal (HPA) axis but too short to induce massive genomic effects of cortisol. Subcellular fractions of thymocytes (cytosol, nucleus, membrane) were investigated using quantitative 2D DIGE with MALDI-TOF/TOF mass spectrometry. In total, 108 proteins with differential subcellular localizations were identified. The specificity of the changes induced by psychological stress was reflected by the prominent modulation of proteins involved in the HPA and sympathoadrenal medullar (SAM) axis such as HMGB1 and NHERF1. Intracellular trafficking was characterized by a dominant protein exodus from the cytosol. Real translocation was observed for 9 proteins with 6 that shuttled from the cytosol to the nucleus (HYOU1, HNRPF, HNRPC, STRAP, PSA1, PPA1) and 3 from the nucleus to the cytosol (HMGB1, NHERF1, PSMA1). Proteins showing subcellular reshuffling were largely involved in transcription and translation processes (39 of 108) with a significant enrichment of RNA splicing factors. Bioinformatics analysis revealed significant enrichment for protein kinase A and 14-3-3 signaling, probably reflecting real non-genomic effects. This is the first study investigating rapid effects of stress-induced HPA activation in vivo at the proteome level.