Pain is the most important clinical feature of acute pancreatitis (AP); however, its specific mechanism is currently unclear. In this study, we showed that AP caused an increase in nitric oxide (NO) secretion, activated the NF-B pathway in the dorsal root ganglia (DRGs), and caused pain. We established an AP model in vivo and tested the expression of NO, the kappa opioid receptor (KOR), and pain factors. We showed that NO in AP was significantly elevated and increased the expression of pain factors. Next, by treating DRGs in vitro, it was found that NO activated the NF-B pathway; conversely, NF-B had no effect on NO. Moreover, inhibition of NF-B promoted the KOR, whereas NF-B did not change after KOR activation. Finally, behavioral experiments showed that a NO donor increased the pain behavior of mice, while a NO scavenger, NF-B inhibitor, or KOR agonist attenuated the pain response in mice. These results suggest that iNOS/NO/NF-B/KOR may be a key mechanism of pain in AP, providing a theoretical basis for the use of peripheral-restricted KOR agonists for pain treatment in AP.Copyright © 2020 Mengwen Xue et al.