Calcium-binding protein S100A9 and its C-terminus peptide (mS100A9p) are anti-inflammatory and induce antinociception in rodents. We investigated the mechanisms involved in this effect, and whether they depend or not on the anti-inflammatory properties of mS100A9p. In mice, mS100A9p inhibited thermal and mechanical hyperalgesia and allodynia induced by either carrageenan or formalin, without interfering with paw edema. mS100A9p also inhibited myeloperoxidase activity (MPO), a marker of granulocyte infiltration, induced by carrageenan, but increased MPO after formalin intraplantar injection. The in vivo analgesic properties of mS100A9p were independent of opioid receptor activation. Calcium flux into dorsal root ganglia neurons induced by KCl was inhibited by mS100A9p, suggesting that this protein is able to inhibit signaling, in sensory neurons. The inhibitory effects of mS100A9p on primary afferent signaling were neither due to intracellular calcium store inhibition nor to calcium chelating properties. However, mS100A9p was able to inhibit calcium currents carried by transiently expressed N-type, but not L-type calcium channels, as demonstrated both by gene transfection techniques and electrophysiology. These data demonstrate that mS100A9p interferes with mechanisms involved in nociception, hyperalgesia and calcium signaling in sensory neurons, modulating primary afferent nociceptive signal by inhibiting activation of N-type voltage operated calcium channels.