The guanylin peptides – guanylin, uroguanylin, and renoguanylin (RGN) – are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study uses one member of these peptides – RGN – to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish (Opsanus beta). Protein kinase G is shown to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation. RGN is shown to reverse the absorptive short-circuit current and increase conductance in the Gulf toadfish intestine. These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel to the apical membrane, which are negated by PKA inhibition. Moreover, RGN decreases HCO3− secretion, likely by limiting the exchange activity of SLC26a6 (a HCO3−/Cl− antiporter), a reduction that is enhanced by PKA inhibition. RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO3− secretion.