Ca²⁺ is the key element in regulated exocytosis controlling multiple steps during the exocytic pre- and post-fusion stages. We have recently described a "fusion-activated“ Ca²⁺-entry (FACE) via vesicular P2X₄ receptors during the post-fusion stage of lamellar body (LB) exocytosis in alveolar type II (ATII) cells. FACE regulates fusion pore expansion and vesicle content release (PNAS, 2011 Aug 30;108(35):14503-8). Yet, the question remained how this locally restricted Ca²⁺ signal is translated into mechanical force promoting fusion pore expansion. In this study we tested whether synaptotagmins could form a molecular link between FACE and fusion pore expansion. Members of the synaptotagmin family that localize to secretory vesicles have been found to promote fusion pore expansion via Ca²⁺ binding to C2 domains. Using RT-PCR and western-blotting we identified synaptotagmin-7 as the Ca²⁺-binding isoform predominantly expressed in primary ATII cells. Immunofluorescence confirmed that synaptotagmin-7 is localised on LBs. In functional studies over-expressing wt or mutant synaptotagmin-7 with impared Ca²⁺-binding we directly assessed dynamic changes in fusion pore diameters by analysing diffusion of fluorescent dyes through the fusion pore. Our results showed that neither wt nor mutant synaptotagmin-7 affects fusion pore expansion in the absence of FACE. Surprisingly, synaptotagmin-7 restricted fusion pore expansion in the presence of FACE. In summary, our results showed that synaptotagmin-7 works as "negative" regulator of LB fusion pore expansion in the presence of Ca²⁺ and hence does not provide the link between FACE and fusion pore expansion in LB exocytosis.