Objective: This study was conducted to investigate effects of the combination of glucagon-like peptide-1 (GLP-1) and fibroblast growth factor 21 (FGF21) on beta-cell function and survival by alleviation of endoplasmic reticulum (ER) stress. Methods: We treated cultured beta-cell lines from mouse and rat (MIN6 and INS-1 cells) with thapsigargin or tunicamycin to induce ER stress. The expression of ER stress markers and glucose-stimulated insulin secretion (GSIS) were measured in the absence or presence of exendin-4 and/or FGF21. The INS-1 cell was treated with thapsigargin or tunicamycin in the absence or presence of exendin-4 and/or FGF21 to evaluate GSIS. To evaluate effects on beta-cell survival, MTT assay was performed and apoptotic and antiapoptotic markers were measured. Results: The expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP was induced by thapsigargin or tunicamycin, but reduced in the presence of exendin-4 or FGF21 in MIN6 and INS-1 cells. In INS-1 cells, GSIS was decreased by thapsigargin or tunicamycin. However, exendin-4 or FGF21 recovered GSIS under ER stress with different glucose concentration. In the MTT assay, the viability of MIN6 and INS-1 cells was reduced by thapsigargin or tunicamycin, but improved in the presence of exendin-4 or FGF21. In INS-1 cells, the expression of Bcl-2 was increased and that of Bax was decreased after treatment of exendin-4 or FGF21. All the results tended to be more effective in the combination of exendin-4 and FGF21 than exendin-4 or FGF21 alone. Conclusion: We demonstrated that the combination of GLP-1 and FGF21 improved beta-cell function and survival in an additive or synergistic fashion. These effects of GLP-1 and FGF21 might be achieved by alleviation of ER stress through different mechanisms.