Foraminifera are commonly used to reconstruct paleoenvironmental conditions based on the taxonomical composition, as well as elemental and/or isotopic signatures of their calcareous tests. A major problem, often referred to as the ‘vital effect’, is that such geochemical signatures stored in inorganic calcium carbonates differ greatly under the same environmental conditions. This effect was previously explained by proportional contributions from passive vs active ion transport patterns, but their details are still investigated. In this study, the functional role of pseudopodial structures during chamber formation is elucidated by detailed observation of Ammonia beccarii (Linnaeus) using a time-lapse optical imaging system and high-resolution electron microscopy. For the first time, we document triple organic layers sandwiching carbonate precipitation sites. The three major organic layers (outer organic layer, primary organic sheet, and inner organic layer) are formed by an initial framework of pseudopodia overlaid with further layer-like pseudopodia. The POS seems to facilitate early calcium carbonate nucleation, then entrapped by double precipitation sites. We further show that calcification starts when outer/inner organic layers still reveal tiny gaps (holes within the framework) that may serve as pathways for passive ion exchange (e.g., Mg 2+ ) between seawater and the confined precipitation space. Nevertheless, the majority of wall thickening occurs when the precipitation site is completely isolated from seawater that implicates of active ion exchange. This may explain the differences in Mg/Ca ratios in early and later stages of calcification observed in previous studies. Our study resolves a key ‘missing piece’ in understanding foraminiferal calcification. The ‘vital effect’ is directly linked to spatio-temporal organization of the ‘biomineralization sandwich’ controlled by the three major organic layers. This study exemplifies the importance of culture experiments and in-depth observations of living organisms in order to interpret and calibrate biogeochemical proxies.