Various preclinical and clinical studies have linked diabetes and breast cancer, but little is known regarding the molecular mechanism involved. This study aimed to investigate the effect of high glucose and insulin in breast cancer cells (MCF-7: non-invasive, hormone dependent, and MDA-MB-231: invasive, hormone independent). In contrast to MCF-7 cells, high glucose augmented proliferation of MDA-MB-231 cells as observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine assays. The high-glucose condition led to increased expression of cyclin D1, de-phosphorylation of p38, and increased phosphorylation of ERK in MDA-MB-231 cells but not in MCF-7 cells. Interestingly, we observed increased phosphorylation of GSK-3β, NF-κB, and ERα only in MCF-7 cells, highlighting their role as potential targets in prevention of progression of breast cancer under a high-glucose and insulin condition. Furthermore, insulin treatment under a high-glucose condition resulted in increased histone H3 phosphorylation and de-acetylation only in MDA-MB-231 cells. Taken together, we provide the first evidence that high glucose and insulin promotes proliferation of MDA-MB-231 cells by differential alteration of GSK-3β, NF-κB, and ERα expression and histone H3 modifications, which may directly or indirectly modulate the expression of genes involved in its proliferation.