The study addressed the functional link between remodelling of the actomyosin cytoskeleton in pancreatic β-cells and the regulation of insulin secretion. Confocal microscopy revealed that myosin heavy chain (MHC) IIA co-localized very well with filamentous (F)-actin in RINm5F cells but MHCIIB did not. Subcellular localization of MHCIIB was not altered by stimulation with 30 mM KCl (which evokes Ca²⁺-dependent insulin secretion). In contrast MHCIIA redistributed in a manner similar to F-actin, especially towards the apical surface, but also away from peripheral regions towards cell contact points on the basal surface. Finally, Ca²⁺-dependent insulin secretion was inhibited by stabilization of actin filaments with jasplakinolide. The results support a role for the MHCIIA/actin cytoskeleton in regulating insulin secretion.