Cholesterol is central to pancreatic β-cell physiology and alterations in its homeostasis contribute to β-cell dysfunction. Several mechanisms regulate its intracellular level, including biosynthesis through the mevalonate pathway and uptake through the low-density lipoprotein receptor (LDLR). Recently, we have shown that proprotein convertase subtilisin/kexin type 9 (PCSK9), expressed in the pancreas, may participate in the control of cholesterol levels by modulating LDLR degradation, and its regulation may influence glucose-stimulated insulin secretion. The aim of this study was to better investigate the action of PCSK9 on the β-cell, highlighting its possible involvement in regulating the architecture, function and crosstalk of intracellular organelles. INS1E cells were transfected with Pcsk9 siRNA or cDNA and their effect on the structure and dynamics of intracellular organelles was assessed by electron microscopy (EM) and super-resolution fluorescence microscopy. EM experiments revealed that both depletion and overexpression of PCSK9 have an impact on the organisation of the early secretory apparatus. In both conditions, enlarged endoplasmic reticulum cisternae, hypertrophic trans-Golgi network and reduced formation and maturation of insulin granules were detected. Consequently, an increase in the proinsulin/insulin ratio was measured. Interestingly, not only changes in the early secretory pathways were observed, but also modifications in the architecture and distribution of several intracellular organelles, supporting the possibility that modulation of PCSK9 may have an impact on their function and crosstalk. Our data point to pancreatic PCSK9 as a new critical regulator of organelle architecture and crosstalk in β-cells and suggest that a homeostatic level of this protein is necessary to preserve β-cell function. Clarifying the mechanisms of action of this protein in the pancreas is essential in order to better exploit the potential of currently available PCSK9 inhibitors and cholesterol-fighting drugs.