Malpighian tubules (MTs) and hindgut constitute the functional kidney of insects. MTs are outpouches of the gut and in most insects demonstrate proximodistal heterogeneity in function. In most insects, such heterogeneity is confined to ion/fluid secretion in the distal portion and ion/fluid reabsorption in the proximal portion. In contrast, MTs of larval Lepidoptera (caterpillars of butterflies and moths), are comprised of five regions that differ in their association with the gut, their structure, and ion/fluid transport function. Recent studies have shown that several regions can rapidly and reversibly switch between ion secretion and reabsorption. The current study employed RNAseq, pharmacology and electrophysiology to characterize four distinct regions of the MT in larval Trichoplusia ni. Luminal microelectrode measurements indicate changes in [K+], [Na+] and pH as fluid passes through different regions of the tubule. In addition, the regions examined differ in gene ontology enrichment, and demonstrate robust gradients in expression of ion transporters and endocrine ligand receptors. Lastly, the study provides evidence for direct involvement of voltage-gated and ligand-gated ion channels in epithelial ion transport of insect MTs.