The Golgi apparatus (GA) is a dynamic intracellular Ca²⁺ store endowed with complex Ca²⁺ homeostatic mechanisms in part distinct from those of the endoplasmic reticulum (ER). We describe the generation of a novel fluorescent Ca²⁺ probe selectively targeted to the medial-Golgi. We demonstrate that in the medial-Golgi: (i) Ca²⁺ accumulation takes advantage of two distinct pumps, the sarco/endoplasmic reticulum Ca²⁺ ATPase and the secretory pathway Ca²⁺ ATPase1; (ii) activation of IP3 or ryanodine receptors causes Ca²⁺ release, while no functional two-pore channel was found; (iii) luminal Ca2+ concentration appears higher than that of the trans-Golgi, but lower than that of the ER, suggesting the existence of a cis- to trans-Golgi Ca²⁺ concentration gradient. Thus, the GA represents a Ca²⁺ store of high complexity where, despite the continuous flow of membranes and luminal contents, each sub-compartment maintains its Ca²⁺ identity with specific Ca²⁺ homeostatic characteristics. The functional role of such micro-heterogeneity in GA Ca²⁺ handling is discussed.