cAMP-responsive element–binding protein (CREB) is required for β-cell survival by regulating expression of crucial genes such as bcl-2 and IRS-2. Using MIN6 cells and isolated rat pancreatic islets, we investigated the signaling pathway that controls phosphorylation and protein level of CREB. We observed that 10 mmol/l glucose–induced CREB phosphorylation was totally inhibited by the protein kinase A (PKA) inhibitor H89 (2 μmol/l) and reduced by 50% with the extracellular signal–regulated kinase (ERK)1/2 inhibitor PD98059 (20 μmol/l). This indicates that ERK1/2, reported to be located downstream of PKA, participates in the PKA-mediated CREB phosphorylation elicited by glucose. In ERK1/2-downregulated MIN6 cells by siRNA, glucose-stimulated CREB phosphorylation was highly reduced and CREB protein content was decreased by 60%. In MIN6 cells and islets cultured for 24–48 h in optimal glucose concentration (10 mmol/l), which promotes survival, blockade of ERK1/2 activity with PD98059 caused a significant decrease in CREB protein level, whereas CREB mRNA remained unaffected (measured by real-time quantitative PCR). This was associated with loss of bcl-2 mRNA and protein contents, caspase-3 activation, and emergence of ultrastructural apoptotic features detected by electron microscopy. Our results indicate that ERK1 and -2 control the phosphorylation and protein level of CREB and play a key role in glucose-mediated pancreatic β-cell survival.