β-adrenergic blockers (β-blockers) are extensively used to inhibit β-adrenoceptor activation and subsequent cAMP production in many cell types. In this study, we characterized the effects of β-blockers on mouse pancreatic β-cells. Unexpectedly, high concentrations (100 μM) of β-blockers (propranolol and bisoprolol) paradoxically increased cAMP levels 5-10 fold, enhanced Ca(2+) influx, and stimulated a 2-4 fold increase in glucose- and glimepiride-induced insulin secretion in MIN6-K8 clonal β-cells and isolated mouse pancreatic islets. These effects were observed despite minimal expression of β-adrenoceptors in these cells. Mechanistically, the cAMP increase led to ryanodine receptor 2 (RYR2) phosphorylation via protein kinase A (PKA), triggering Ca(2+)-induced Ca(2+) release (CICR). CICR then activates transient receptor potential cation channel subfamily M member 5 (TRPM5), resulting in increased Ca(2+) influx via voltage-dependent Ca(2+) channels. These effects contradict the conventional understanding of the pharmacology of β-blockers, highlighting the variability in β-blocker actions depending on the experimental context.