Pancreatic β-cells regulate glucose homeostasis by secreting insulin in response to glucose elevation and G protein-coupled receptor (GPCR) activation. Neuropeptide Y (NPY) and somatostatin (SST) attenuate insulin secretion through G_i activation of Y₁ and SSTR_1&5 receptors, respectively. The downstream pathways altered by NPY and SST are poorly understood. Thus, we investigated these underlying mechanisms. NPY and SST increase cellular redox potential, suggesting that their inhibitory effect may not be mediated through metabolic inhibition. NPY does not affect intracellular calcium ([Ca²⁺]_i) activity upon glucose stimulation, whereas SST alters this response. G_βγ-subunit inhibition by gallein attenuates insulin secretion but does not alter metabolism or [Ca²⁺]_i. mSIRK-induced G_βγ activation does not modulate glucose metabolism but increases [Ca²⁺]_i activity and potentiates insulin release. Cotreatment with gallein and NPY or SST reduces insulin secretion to levels similar to that of gallein alone. mSIRK and NPY cotreatment potentiates insulin secretion similarly to mSIRK alone, whereas mSIRK and SST treatment decreases insulin release. The data support a model where SST attenuates secretion through G_βγ inhibition of Ca²⁺ activity, while NPY activates a Ca²⁺-independent pathway mediated by G_α. GPCR ligands signal through multiple pathways to inhibit insulin secretion, and determining these mechanisms could lead to novel diabetic therapies.