Background: It has been demonstrated that kappa-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the kappa-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how kappa-opioid receptors respond to hypoxia and reoxygenation. Methods: Mouse neuroblastoma Neuro2A cells were stably transfected with mouse kappa-opioid receptor-tdTomato fusion protein or Flag-tagged mouse kappa-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the kappa-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of kappa-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. Results: Hypoxic conditions caused the kappa-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific kappa-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific kappa-opioid receptor. kappa-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular kappa-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced kappa-opioid receptor internalization. Conclusions: Hypoxia induces reversible kappa-opioid receptor internalization, which was inhibited by selective kappa-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between kappa-opioid receptor and hypoxia via kappa-opioid receptor activation and the dynamin-dependent mechanism.