The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute posttraumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in "cation-free" solution and blocked by the nonspecific Cl-channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-β-S. Notably, S1P3 receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P3-/-mice, whereas in control S1P1 floxed (S1P1 f1/f1) mice and mice with a nociceptor-specific deletion of S1P1 -/- receptor (SNS-S1P1 -/-), neither the S1P-induced responses in vitro nor the S1Pevoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-proteindependent activation of an excitatory Cl- conductance that is largely mediated by S1P3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.