Visceral painful stimulation induces spinal neuronal plasticity changes and intracellular c-AMP-dependent protein kinase (PKA)-mediated signaling cascades may contribute to this nociceptive processing at the spinal cord level. Previous clinical studies indicated that neurosurgical interruption of post-synaptic dorsal column (PSDC) pathway by performing midline myelotomy could effectively alleviate intractable visceral pain in patients with severe cancer pain. However, the role of PKA-mediated visceral nociception mechanisms in PSDC neurons was less known. In the current study, we used multiple experimental approaches to investigate the involvement of PKA in nociceptive signaling in the spinal cord and PSDC neurons in a rat model of visceral pain induced by intracolonic injection of mustard oil. The results showed that mustard oil injection significantly changed the exploratory behavioral activity in rats in terms of decreased numbers of entries, traveled distance, active and rearing time, rearing activity, and increased resting time. Intrathecal administration of a PKA inhibitor, H 89 partially reversed the visceral pain-induced exploratory behavioral effects. Western blot studies showed that mustard oil injection significantly induced the expression of PKA protein in the lumbosacral spinal cord. Immunofluorescent staining in pre-labeled PSDC neurons showed that visceral pain greatly induced the profile numbers of neuron expressing PKA. Finally, we also found that the PKA inhibitor, H89 significantly reduced the visceral pain-induced expressions of c-fos and phosphorylated c-AMP –responsive element binding (CREB) protein in spinal cord. The results of this study suggest that the PKA signaling cascade may contribute to visceral nociceptive neurotransmission in spinal PSDC pathways. (NIH DE15814, NS40723)