The Golgi apparatus (GA) is a dynamic store of Ca2+ that can be released into the cell cytosol. It can thus participate in the regulation of the Ca2+ concentration in the cytosol ([Ca2+]cyt), which might be critical for intra-Golgi transport. Secretory pathway Ca2+-ATPase pump type 1 (SPCA1) is important in Golgi homeostasis of Ca2+. The subcellular localization of SPCA1 appears to be GA specific, although its precise location within the GA is not known. Here, we show that SPCA1 is mostly excluded from the cores of the Golgi cisternae and is instead located mainly on the lateral rims of Golgi stacks, in tubular noncompact zones that interconnect different Golgi stacks, and within tubular parts of the trans Golgi network, suggesting a role in regulation of the local [Ca2+]cyt that is crucial for membrane fusion. SPCA1 knockdown by RNA interference induces GA fragmentation. These Golgi fragments lack the cis-most and trans-most cisternae and remain within the perinuclear region. This SPCA1 knockdown inhibits exit of vesicular stomatitis virus G-protein from the GA and delays retrograde redistribution of the GA glycosylation enzymes into the endoplasmic reticulum caused by brefeldin A; however, exit of these enzymes from the endoplasmic reticulum is not affected. Thus, correct SPCA1 functioning is crucial to intra-Golgi transport and maintenance of the Golgi ribbon.