Pancreatic islets are susceptible to hypoxia during isolation, culture, and transplantation. An intervention that protects against hypoxia while preserving β-cell function will improve islet quality. Adrenaline suppresses insulin secretion and oxygen consumption in pancreatic islets. These findings suggest that adrenergic action creates metabolic quiescence in β-cells. Our objective was to determine whether adrenaline is protective during hypoxic culture in an insulinoma cell line. Min6 cells were cultured for 72 h in four groups (n=3/group): normoxia, normoxia+100 nM adrenaline, hypoxia (1% O2), hypoxia+100 nM adrenaline. Adrenaline was removed for 1 h or 48 h before functional assessments. Stimulated insulin release was determined in 16.7 mM glucose and fold change calculated from 0 mM glucose. RT2-PCR hypoxia arrays were used to determine mRNA expression. PCR arrays confirmed Min6 cells in 1% O2 have a hypoxic gene signature. Immediately after treatment, adrenaline supplementation increased stimulated insulin concentrations in hypoxic (40.6±5.3 ng/ngDNA) and normoxic (39.4±3.8 ng/ngDNA) cultures compared to normoxic (14.1±1.2 ng/ngDNA) and hypoxic cultures (15.2±1.1 ng/ngDNA). After 48 h recovery, stimulated insulin concentrations were increased 46±7% (P<0.05) in normoxia+adrenaline compared to normoxia. Hypoxia reduced (P<0.05) glucose stimulated insulin secretion to 1.8±0.3 fold compared to normoxia at 3.4±0.6 fold, but hypoxia+adrenaline (2.6±0.4 fold) was similar to normoxia. Oxygen consumption rate was not different between the groups, but glucose-stimulated NADH concentrations were greater in normoxic cells. Hypoxia and adrenaline reduced cell density compared to normoxia and the number of non-adhered cells was similar in all conditions, indicating slower replication rates, not cell death. Adrenaline supplementation to hypoxic β-cell cultures preserved insulin secretion. Although adrenaline reduced proliferation of insulinoma cells, the impact will likely be less in mature islets because viability was maintained. These data identify the potential use of adrenergic agonists as a protective agent against hypoxia during islet isolations procedures to improve β-cell viable and function.