Morphine is the most commonly used and most effective analgesic in the clinic.However, its use is limited by the tolerance.Evidence indicates that the δ-opioid receptor (DOR) is essential for morphine antinociceptive tolerance;however, their underlying mechanisms are poorly understood.Here, we show that cyclin-dependent kinase 5 (Cdk5), activated in morphine antinociceptive tolerance, directly phosphorylates DOR at Thr-161 in DRG neurons.Our studies indicated that inhibition of Cdk5 activity or overexpression of a DOR mutant lacking the Cdk5 phosphorylation site displayed relatively low cell surface expression and relatively low abilities to form heterodimers of DOR and MOR;intrathecal delivery of a construct expressing the T161A mutant of DOR or Tat-DOR-2L peptide attenuated morphine antinociceptive tolerance in normal and inflammatory pain rats.The present study indicates that Cdk5-mediated phosphorylation of DOR at Thr-161 plays a crucial role in the development of morphine tolerance and suggests the possibility of targeting DOR phosphorylation at Thr-161 to attenuate morphine tolerance during pain management.