Rhodnius prolixus is a blood-feeding insect vector of Chagas’ disease. During each blood meal the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These large blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared to the insect’s haemolymph. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin and the effect of the neuropetide modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated transepithelial short circuit current (Isc) was blocked by treatment with the Na+ channel blocker amiloride or the Na+/K+-ATPase inhibitor ouabain. Incubation in Na+-free, but not Cl–free saline, blocked the effect of serotonin on Isc. Moreover, treatment with NKCC and NCC blockers had no effect on Isc or fluid secretion. Treatment with neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na+ undergoes active transport via an apical amiloride-sensitive Na+ channels and a basolateral ouabain-sensitive Na+/K+-ATPase while Cl- is transported through passive paracellular pathway. RhoprCCHamide2 reduces transport by inhibiting transcellular Na+ transport.