14-3-3 proteins are highly conserved across eukaryotes, typically encoded by multiple genes in most species. Drosophila has only two such genes, 14-3-3zeta (leo), encoding two isoforms LEOI and LEOII, and 14-3-3epsilon. We report a bona fide third functional isoform encoded by leo divergent from the other two in structurally and functionally significant areas, thus increasing 14-3-3 diversity in Drosophila. Furthermore, we used a novel approach of spatially restricted leo abrogation by RNA-interference and revealed differential LEO distribution in adult heads, with LEOIII enrichment in neurons essential for learning and memory in Drosophila.