Neutrophils play a fundamental role in host defense by neutralizing pathogens through the generation of ROS by NOX2. In nonexcitable cells, Ca(2+) influx is essentially mediated via SOCE, a complex mechanism in which depletion of intracellular Ca(2+) stores from the ER results in Ca(2+) entry through Ca(2+) SOCs at the plasma membrane. In this regard, it is well established that extracellular Ca(2+) entry participates to NOX2 activation. S1P, produced by SphKs, has been involved in Ca(2+) homeostasis and thus, could intervene in NOX2 regulation. The aim of this study was to characterize the importance of SphKs in NOX2 activation and the signaling cascade involved in this mechanism. Treatment of neutrophil-like dHL-60 cells by DHS, a SphK inhibitor, and SphK siRNA inhibited fMLF-induced NOX2 activity. Sequential activation of cells by thapsigargin and the phorbol ester PMA revealed that SphK-regulated NOX2 activity relies on intracellular Ca(2+) store depletion. Confocal microscopy and immunoblot analysis showed that stimulation by thapsigargin and PMA mediated S100A8/A9 recruitment to the plasma membrane and p38 MAPK activation. S100A8/A9 translocation decreased when SphK activity was blocked. This result was confirmed in purified human neutrophils, which were physiologically stimulated by fMLF. In addition, p38 MAPK was found to be regulated by SphKs. These results define a pathway leading to NOX2 activation, in which p38 MAPK-mediated S100A8/A9 translocation is regulated by Ca(2+) store depletion-dependent SphK activation.