Calnuc is an ubiquitous, EF-hand Ca²⁺ binding protein found in the cytoplasm where it binds to Gi3, within the Golgi lumen where it constitutes a Ca²⁺ storage pool, and secreted outside the cell. Here we investigated the pathway of secretion of calnuc in AtT20 cells. We found by pulse-chase experiments that calnuc is synthesized in the endoplasmic reticulum, transported to the Golgi where it remains 12 h and undergoes posttranslational modification (O-glycosylation and sulfation) followed by secretion into the culture medium. We examined if calnuc is secreted by the constitutive or regulated secretory pathway in AtT20 cells. By immunofluorescence and immunogold labeling, endogenous calnuc is found in immature secretion granules (ISG) but not mature regulated secretory granules (RSG), whereas overexpressed calnuc-GFP is found in both ISG and RSG where it colocalizes with ACTH. Neither calnuc nor calnuc-GFP are released by the regulated secretory pathway, suggesting that endogenous calnuc and calnuc-GFP are progressively removed from ISG and RSG during granule maturation. We conclude that calnuc is secreted via the constitutive-like pathway and represents a useful endogenous marker for this pathway in AtT20 cells. Together, these observations indicate that calnuc has a unique itinerary as it is retained in the Golgi and is then constitutively secreted extracellularly where it may influence cell behavior via its Ca²⁺ -binding properties.