14-3-3 is a family of proteins comprising several isoforms that, in many cases, promote cell survival by association with proapoptotic proteins. This study was designed to obtain further understanding of the 14-3-3 role in apoptosis regulation, by analyzing apoptosis-related protein-14-3-3 interactions. Western blot analysis of an eluted fraction from the 14-3-3-affinity chromatography column identified proapoptotic proteins as receptor-interacting protein 3 and Bcl-2-antagonist/killer as new phophorylation-dependent 14-3-3-binding proteins under physiological conditions. The apoptosis inducer C2-ceramide promoted decay of the 14-3-3-binding signal of protein cell extracts. Investigation of the role of 14-3-3 in C2-ceramide-induced apoptosis showed that depletion of the 14-3-3zeta isoform sensitized to cell death, whereas overexpression of this isoform delayed cell death. A combination of tandem affinity purification and liquid chromatography-tandem MS techniques identified 15 proteins involved in cell survival processes whose 14-3-3-binding status changed during C2-ceramide-induced apoptosis. Under physiological conditions, desmin was clearly identified as a new 14-3-3-interactor protein, and vasodilator-stimulated phosphoprotein, nucleophosmin and calmodulin, whose 14-3-3 binding was suggested by others on the basis of MS analysis, were confirmed here as phosphorylation-dependent 14-3-3-associated proteins. Interestingly, proteins related to the regulation of DNA double-strand break repair in the early stages of apoptosis, such as DNA-dependent protein kinase, or the regulation of cell shrinkage during apoptosis, such as vasodilator-stimulated phosphoprotein and death promoters like receptor-interacting protein 3, were identified as 14-3-3-associated proteins whose 14-3-3-binding status changed when apoptosis was initiated. The functional diversity of these identified proteins suggests that 14-3-3 may regulate the apoptotic process through new mechanisms, in addition to others previously characterized.