There is a correlation between circadian disruption, type 2 diabetes mellitus (T2DM), and islet failure. However, the mechanisms underlying this association are largely unknown. Pancreatic islets express self-sustained circadian clocks essential for proper β-cell function and survival. We hypothesized that exposure to environmental conditions associated with disruption of circadian rhythms and susceptibility to T2DM in humans disrupts islet clock and β-cell function. To address this hypothesis, we validated the use of Per-1:LUC transgenic rats for continuous longitudinal assessment of islet circadian clock function ex vivo. Using this methodology, we subsequently examined effects of the continuous exposure to light at night (LL) on islet circadian clock and insulin secretion in vitro in rat islets. Our data show that changes in the light-dark cycle in vivo entrain the phase of islet clock transcriptional oscillations, whereas prolonged exposure (10 weeks) to LL disrupts islet circadian clock function through impairment in the amplitude, phase, and interislet synchrony of clock transcriptional oscillations. We also report that exposure to LL leads to diminished glucose-stimulated insulin secretion due to a decrease in insulin secretory pulse mass. Our studies identify potential mechanisms by which disturbances in circadian rhythms common to modern life can predispose to islet failure in T2DM.