Objective  To explore the neuronal properties of the pathway from the medial prefrontal cortex (mPFC) to the paraventricular thalamic nucleus of (PVT) and to investigate the effect of modulation of the pathway on physiological pain and acute pain in mice. Methods  CTb was injected into the PVT of GAD67-GFP transgenic mice, and the properties of mPFC neurons projected to PVT were observed. The mPFC-PVT pathway was activated or inhibited by chemogenetics to observe the effects on physiological pain, such as mechanical pain, thermal pain, cold pain, and on capsaicin induced inflammatory pain. Results  CTB-labeled neurons in the mPFC were mainly distributed in layer V and layer VI, and were not double-labeled with GAD67-GFP. Chemogenetic activation of the mPFC-PVT pathway significantly decreased the mechanical pain threshold (p < 0.0001) and shortened the thermal pain latency (p < 0.001), but had no obvious effects on cold pain. Inhibition of this pathway significantly increased the mechanical pain threshold (p < 0.05). Activation of the pathway increased the paw licking time (p < 0.05) in acute inflammatory pain induced by intraplantar injection of capsaicin. Conclusion  mPFC-PVT pathway is a non GABAergic projection and its activation can promote mechanical pain, thermal pain, and acute inflammatory pain in mice.