The mammalian Golgi complex consists of stacks of cisternae linked laterally into a continuous perinuclear ribbon structure. Protein kinase A is stably associated with the Golgi complex during interphase. To analyze its role in Golgi structural maintenance cells were depleted of protein kinase A regulatory subunits using small interfering RNAs. Under these conditions, the catalytic subunits redistributed to the cytosol and the entire Golgi complex underwent disassembly into multiple juxtanuclear fragments. A similar effect took place following pharmacological inhibition or redistribution of the complete holoenzyme to the cytosol. Golgi fragments maintained their polarization and competence for anterograde protein trafficking. By electron microscopy, they were identified as whorl-like structures composed of concentrically arrayed cisternae. To test a possible role of protein kinase A in Golgi biogenesis we analyzed its involvement during Golgi reassembly from the endoplasmic reticulum. In cells incubated with protein kinase A inhibitors, Golgi reconstruction was arrested at a late step of the reassembly process. This is consistent with the stage of enzyme recruitment from cytosol to emerging Golgi membranes during the reassembly process. We conclude that protein kinase A activity plays a relevant role in the assembly and maintenance of a continuous Golgi ribbon from separated membrane stacks.