The mechanism of action of acetaminophen is currently widely discussed. Direct inhibition of cyclooxygenase isoforms remains the commonly advanced hypothesis. We combined behavioral studies with molecular techniques to investigate the mechanism of action of acetaminophen in a model of tonic pain in rats. We show that acetaminophen indirectly stimulates spinal 5-hydroxytryptamine (5-HT)(1A) receptors in the formalin test, thereby increasing transcript and protein levels of low-affinity neurotrophin receptor, insulin-like growth factor-1 (IGF-1) receptor alpha subunit, and growth hormone receptor and reducing the amount of somatostatin 3 receptor (sst3R) mRNA. Those cellular events seem to be important for the antinociceptive activity of acetaminophen. Indeed, down-regulation of sst3R mRNA depends on acetaminophen-elicited, 5-HT1A receptor-dependent increase in neuronal extracellular signal-regulated kinase 1/2 (ERK1/2) activities that mediate antinociception. In addition, spinal growth hormone (GH) and IGF-1 receptors would also be involved in the antinociceptive activity of the analgesic at different degrees. Our results show the involvement of specific 5-HT1A receptor-dependent cellular events in acetaminophen-produced antinociception and consequently indicate that inhibition of cyclooxygenase activities is not the exclusive mechanism involved. Furthermore, we propose that the mechanisms of 5-HT1A receptor-elicited antinociception and the role of the spinal ERK1/2 pathway in nociception are more intricate than suspected so far and that the GH/IGF-1 axis is an interesting new player in the regulation of spinal nociception.