Each of the lateral capsular division of central nucleus of amygdala(CeLC), periaqueductal gray (PAG), rostral ventromedial medulla(RVM) and spinal cord has been proved to contribute to the development of opioid-induced hyperalgesia(OIH). Especially, Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII alpha) in CeLC and spinal cord seems to play a key role in OIH modulation. However, the pain pathway through which CaMKII alpha modulates OIH is not clear. The pathway from CeLC to spinal cord for this modulation was explored in the present study. Mechanical and thermal hyperalgesia were tested by von Frey test or Hargreaves test, respectively. CaMKII alpha activity (phospho-CaMKII alpha, p- CaMKII alpha) was evaluated by western blot analysis. CaMKII alpha antagonist (KN93) was micro-infused into CeLC, spinal cord or PAG, respectively, to evaluate its effect on behavioral hyperalgesia and p-CaMKII alpha expression in CeLC, PAG, RVM and spinal cord. Then the underlying synaptic mechanism was explored by recording miniature excitatory postsynaptic currents (mEPSCs) on PAG slices using whole-cell voltage-clamp methods. Results showed that inhibition of CeLC, PAG or spinal CaMKII alpha activity respectively by KN93, reversed both mechanical and thermal hyperalgesia. Microinjection of KN93 into CeLC decreased p- CaMKII alpha expression in CeLC, PAG, RVM and spinal cord; while intrathecal KN93 can only block spinal but not CeLC CaMKIIa activity. KN93 injected into PAG just decreased p-CaMKII alpha expression in PAG, RVM and spinal cord, but not in the CeLC. Similarly, whole-cell voltage-clamp recording found the frequency and amplitude of mEPSCs in PAG cells were decreased by KN93 added in PAG slice or micro-infused into CeLC in vivo. These results together with previous findings suggest that CaMKII alpha may modulate OIH via a CeLC-PAG-RVM-spinal cord descending facilitative pain pathway.