Lipotoxicity is considered one of the main causes of deterioration in β-cells function. Glucagon-like peptide-1 (GLP-1) has been revealed to protect and improve pancreatic β-cell function against lipotoxicity. However, the mechanism behind these is largely unknown. The aim of this study was to investigate the effects of GLP-1 on cholesterol-induced lipotoxicity in INS-1 cells and examine the underlying mechanisms. The cell viability was determined, and caspase-3 was used to assess the effects of GLP-1 on cholesterol-induced apoptosis. The alterations of miR-27a and ABCA1 resulting from incubation with cholesterol or GLP-1 were detected by real-time PCR and western blot. The inhibition and overexpression of miR-27a were established to explore the effects of a GLP-1-mediated decrease in miR-27a. Further, Oil red O staining and cholesterol measurement were used to detect lipid accumulation. The β-cells function was measured in glucose-stimulated insulin secretion. Our data shows that cholesterol significantly attenuated cell viability, promoted cell apoptosis, facilitated lipid accumulation, and impaired β-cells function, and these effects were significantly reversed by GLP-1. Furthermore, the results demonstrated that GLP-1 decreased miR-27a expression and increased the expression of ABCA1. In conclusion, GLP-1 may affect cholesterol accumulation and β-cells dysfunction by regulating the expression of miR-27a and ABCA1.