Type 2 diabetes (T2D) cases are growing throughout the world. A key characteristic of T2D is damage to the beta cell. This damage affects the beta cell's ability to sense glucose and release insulin in response to elevated blood glucose levels. Nkx6.1 is a beta cell transcription factor essential for differentiation, proliferation, and insulin secretion. To test the effect of hyperglycemia on the beta cell, INS-1 832/13 beta cells were cultured under hyperglycemic conditions for various time durations. This treatment showed that after 12 hours, there was a decrease in Nkx6.1 protein. Even with this decrease in protein levels, there is no change in transcription, colocalization or degradation of Nkx6.1. It is likely that decreased translation at 12 hours causes Nkx6.1 protein levels to drop. Nkx6.1 protein levels remain decreased at 24 hours. Nkx6.1 mRNA decreases, with changes to translation, translocation and degradation at 24 hours. These mechanisms were also validated in rat islets. Understanding the effect of hyperglycemia on Nkx6.1 is imperative to the future development of gene therapies used to treat diabetes.