All forms of diabetes share the common etiology of impaired pancreatic β-cell insulin release to meet the high demands of peripheral tissues. In pancreatic β-cells, mitochondria serve to integrate the metabolism of exogenous nutrients into energy output, which eventually leads to insulin release. As such, mitochondrial dysfunction underlies β-cell failure and the development of diabetes. Mitochondrial regulation of β-cell function occurs through many diverse pathways, including metabolic coupling, maintenance of mitochondria mass, generation of reactive oxygen species , and through interaction with other cellular organelles. In this chapter, we will examine the factors responsible for mitochondrial biogenesis and degradation and their roles in the balance of mitochondrial mass in β-cells. Furthermore, we will focus on the importance of enzymatic regulators of mitochondrial fuel metabolism and control of mitochondrial mass to pancreatic β-cell function, describing how defects in these pathways ultimately lead to diabetes. Clarifying the causes of β-cell mitochondrial dysfunction may inform new approaches to treat underlying etiologies of diabetes.