Rationale: Ca ²⁺ signaling is a key and ubiquitous actor of cell organization and its modulation controls many cellular responses. SERCAs (sarco-endoplasmic reticulum Ca ²⁺ -ATPases) pump Ca ²⁺ into internal stores that play a major role in the cytosolic Ca ²⁺ concentration rise upon cell activation. Platelets exhibit 2 types of SERCAs, SERCA2b and SERCA3 (SERCA3 deficient mice), which may exert specific roles, yet ill-defined. We have recently shown that Ca ²⁺ mobilization from SERCA3-dependent stores was required for full platelet activation in weak stimulation conditions. Objective: To uncover the signaling mechanisms associated with Ca ²⁺ mobilization from SERCA3-dependent stores leading to ADP secretion. Methods and Results: Using platelets from wild-type or Serca3 -deficient mice, we demonstrated that an early (within 5–10 s following stimulation) secretion of ADP specifically dependent on SERCA3 stored Ca ²⁺ is exclusively mobilized by nicotinic acid adenosine dinucleotide-phosphate (NAADP): both Ca ²⁺ mobilization from SERCA3-dependent stores and primary ADP secretion are blocked by the NAADP receptor antagonist Ned-19, and reciprocally both are stimulated by permeant NAADP. In contrast, Ca ²⁺ mobilization from SERCA3-dependent stores and primary ADP secretion were unaffected by inhibition of the production of IP3 (inositol-1,4,5-trisphosphate) by phospholipase-C and accordingly were not stimulated by permeant IP3. Conclusions: Upon activation, an NAADP/SERCA3 Ca ²⁺ mobilization pathway initiates an early ADP secretion, potentiating platelet activation, and a secondary wave of ADP secretion driven by both an IP3/SERCA2b-dependent Ca ²⁺ stores pathway and the NAADP/SERCA3 pathway. This does not exclude that Ca ²⁺ mobilized from SERCA3 stores may also enhance platelet global reactivity to agonists. Because of its modulating effect on platelet activation, this NAADP-SERCA3 pathway may be a relevant target for anti-thrombotic therapy. Graphic Abstract: A graphic abstract is available for this article.