Atrial natriuretic peptide (ANP) and its guanylyl cyclase-A (GC-A) receptor are being involved in metabolism, although their role in the endocrine pancreas is still greatly unknown. The aim of this work is to study a possible role for the ANP/GC-A system in modulating pancreatic beta-cell function. The results presented here show a direct effect of the GC-A receptor in regulating glucose-stimulated insulin secretion (GSIS) and beta-cell mass. GC-A activation by its natural ligand, ANP, rapidly blocked ATP-dependent potassium (K(ATP)) channel activity, increased glucose-elicited Ca(2+) signals, and enhanced GSIS in islets of Langerhans. The effect in GSIS was inhibited in islets from GC-A knockout (KO) mice. Pancreatic islets from GC-A KO mice responded to increasing glucose concentrations with enhanced insulin secretion compared with wild type (WT). Remarkably, islets from GC-A KO mice were smaller, presented lower beta-cell mass and decreased insulin content. However, glucose-induced Ca(2+) response was more vigorous in GC-A KO islets, and basal K(ATP) channel activity in GC-A KO beta-cells was greatly diminished compared with WT. When protein levels of the two K(ATP) channel constitutive subunits sulfonylurea receptor 1 and Inward rectifier potassium channel 6.2 were measured, both were diminished in GC-A KO islets. These alterations on beta-cell function were not associated with disruption of glucose tolerance or insulin sensitivity in vivo. Glucose and insulin tolerance tests were similar in WT and GC-A KO mice. Our data suggest that the ANP/GC-A system may have a modulating effect on beta-cell function.