Members of the conserved 14-3-3 protein family spontaneously self-assemble as homo- and heterodimers via conserved sequences in the first four (alphaA-alphaD) of the nine helices that comprise them. Dimeric 14-3-3s bind conserved motifs in diverse protein targets involved in multiple essential cellular processes including signaling, intracellular trafficking, cell cycle regulation, and modulation of enzymatic activities. However, recent mostly in vitro evidence has emerged, suggesting functional and regulatory roles for monomeric 14-3-3s. We capitalized on the simplicity of the 14-3-3 family in Drosophila to investigate in vivo 14-3-3zeta monomer properties and functionality. We report that dimerization is essential for the stability and function of 14-3-3zeta in neurons. Moreover, we reveal the contribution of conserved amino acids in helices A and D to homo- and heterodimerization and their functional consequences on the viability of animals devoid of endogenous 14-3-3zeta. Finally, we present evidence suggesting endogenous homeostatic adjustment of the levels of the second family member in Drosophila, D14-3-3epsilon, to transgenic monomeric and dimerization-competent 14-3-3zeta.