Both the rate of overall translation and the specific acceleration of proinsulin synthesis are known to be glucose-regulated processes in the β-cell. In this study, we propose that glucose-induced stimulation of overall translation in β-cells depends on a protein phosphatase-1-mediated decrease in serine-51 phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a pivotal translation initiation factor. The decrease was rapid and detectable within 15 min and proportional to the range of glucose concentrations that also stimulate translation. Lowered net eIF2α phosphorylation was not associated with a detectable decrease in activity of any eIF2α kinase. Moreover, okadaic acid blocked glucose-induced eIF2α dephosphorylation, suggesting that the net effect was mediated by a protein phosphatase. Experiments with salubrinal on intact cells and nuclear inhibitor of protein phosphatase-1 (PP1) on cell extracts suggested that this phosphatase was PP1. The net effect contained, however, a component of glucose-induced folding load in the endoplasmic reticulum because coincubation with cycloheximide further amplified the effect of glucose on eIF2α dephosphorylation. Thus, the steady-state level of eIF2α phosphorylation in β-cells is the result of a balance between folding-load-induced phosphorylation and PP1-dependent dephosphorylation. Because defects in the pancreatic endoplasmic reticulum kinase-eIF2α signaling system lead to β-cell failure and diabetes, deregulation of the PP1 system could likewise lead to cellular dysfunction and disease.