Impaired function and decreasing numbers of pancreatic β cells are key factors in the development of type 2 diabetes. This study investigated whether 2,7″-phloroglucinol-6,6′-bieckol protects INS-1 cells against high glucose-induced glucotoxicity and apoptosis. High glucose (30 mM) treatment led to glucotoxicity and induced apoptosis in INS-1 cells. However, treatment with 10–50 μM 2,7″-phloroglucinol-6,6′-bieckol significantly reduced glucotoxicity (22.4%) and increased cell viability (up to 80.9%). Treatment with 2,7″-phloroglucinol-6,6′-bieckol caused a dose-dependent decrease in intracellular reactive oxygen species, lipid peroxidation, and nitric oxide levels, which are increased in INS-1 cells exposed to high glucose. The level of intracellular reactive oxygen species, lipid peroxidation, and nitric oxide levels in INS-1 cells treated with 30 mM glucose and 50 μM 2,7″-phloroglucinol-6,6′-bieckol (101.9%, 0.11 % and 102.1%) was similar to that of the control group (100%). Furthermore, 2,7″-phloroglucinol-6,6′-bieckol significantly reduced the levels of pro-apoptotic Bax, caspase 9, and caspase 3 proteins, and increased the levels of the anti-apoptotic Bcl-2 protein and poly ADP-ribose polymerase. The Bcl-2/Bax ratio was 6-fold higher in 50 μM 2,7″-phloroglucinol-6,6′-bieckol-treated INS-1 cells than in untreated cells. Apoptotic cells were identified using annexin V/propidium iodide staining, and cells treated with 50 μM 2,7″-phloroglucinol-6,6′-bieckol, the numbers of late apoptotic cells decreased over 2-fold compared to that observed in cells treated with 30 mM glucose. Overall, these results suggest that 2,7″-phloroglucinol-6,6′-bieckol has the potential for use as a pharmaceutical agent to protect pancreatic β cells against high glucose-induced apoptosis.