Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and Delta ctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the Delta ctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria.