Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. In this work, we have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. We highlighted the conservation of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding our knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.