The insulin gene is expressed exclusively in the beta cells of the islet of Langerhans. The release of this polypeptide hormone into the bloodstream, principally in response to elevated glucose levels, is essential for controlling carbohydrate metabolism in peripheral tissues. A fundamental cause of diabetes, a disease that affects millions of people and is a major cause of morbidity and mortality, is the inability of beta cells to produce sufficient amounts of insulin, resulting in hyperglycemia. A large effort is underway to identify and characterize the transcriptional regulators of genes, like insulin, that are important in islet beta cell function. It is hoped that this knowledge will provide information into how beta cell function is disrupted in type 2 diabetic individuals, and to provide a foundation for cell-based therapies that may be effective in diabetes treatment. Many of the cis-acting sequences, essential in directing both selective and glucose-inducible transcription within the 5′-flanking region of the insulin gene, have been defined and several of the key trans-activators isolated, including PAX-6, PDX-1, MafA, and BETA2/NeuroD1. In addition, the inactivation of genes encoding these regulatory proteins in mice has established that most play a role in islet cell differentiation during pancreas development. In this review, the regulatory role of the islet-enriched transcription factors of the insulin gene will be discussed, with a focus on their role in adult beta cell function.