Serotonin [5-hydroxytryptamine (5-HT)] released from mast cells or platelets in peripheral tissues is one of the important inflammatory mediators in pain and hyperalgesia. The involvement of 5-HT in pain is complex because it could inhibit or facilitate nociceptive transmission, reflecting the presence of multiple 5-HT subtype receptors on peripheral and central nociceptors. The present study aimed to investigate the involvement of 5-HT(2B) in 5-HT-induced pain and whether the subtype exists in dorsal root ganglion (DRG) neurons. Injecting the 5-HT or 5-HT(2) agonist in hindpaws of mice induced significant hyperalgesia to mechanical stimuli, which was inhibited by the 5-HT(2B/2C) antagonist but not by 5-HT(1A), 5-HT(2A), or 5-HT(3A) antagonists. Therefore, 5-HT(2B) or 5-HT(2C) may be involved in 5-HT-induced mechanical hyperalgesia. The 5-HT(2B/2C) antagonist also blocked 5-HT-induced transient [Ca(2+)] signaling in DRG neurons. All subtypes of 5-HT receptors except 5-HT(2C) and 5-HT(6) are present in DRGs. In situ hybridization also demonstrated 5-HT(2B) mainly expressed in small-to medium-diameter DRG neurons that respond to pain. Likely, 5-HT(2B) mediates 5-HT-induced mechanical hyperalgesia in mice.