Loss of the epithelial intermediate filament protein keratin 8 (K8) in murine β cells leads to irregular insulin vesicles and decreased insulin levels. Because mitochondria are central in glucose-stimulated insulin secretion, the relationship between keratins and β-cell mitochondrial function and morphology was investigated. β cells in murine K8-knockout (K8-/-) islets of Langerhans have increased numbers of mitochondria, which are rounder and have diffuse cristae, as seen by electron microscopy. The mitochondrial network in primary cultured K8-/- β cells is more fragmented compared with K8+/+ mitochondria, correlating with decreased levels of mitofusin 2 and the mitofusin 2- and keratin-binding protein trichoplein. K8-/- β-cell mitochondria have decreased levels of total and mitochondrial cytochrome c, which correlates with a reduction in electron transport complexes I and IV. This provokes loss of mitochondrial membrane potential and reduction of ATP and insulin amount, as seen in K8-/- β cells. Mitochondria in K8 wild-type β cells and MIN6 insulinoma cells overexpressing K8 and 18 are more stationary compared with mitochondria in keratin-deficient cells. In conclusion, keratins, likely through trichoplein-mitofusin interactions, regulate both structural and dynamic functions of β-cell mitochondria, which could have implications for downstream insulin secretion.