Morphine-induced scratching (MIS) is a common adverse effect associated with the use of morphine as analgesia after surgery. However, the treatment of MIS is less than satisfactory due to its unclear mechanism, which needs to be enunciated. We found that intrathecal (i.t.) injections of morphine significantly enhanced scratching behavior in C57BL/6J male mice as well as increased the expressions of protein kinase C β (PKCβ), phosphorylated p38 mitogen-activated protein kinases (MAPK), and ionized calciumbinding adapter molecule 1 (Iba1) within spinal cord dorsal horn. Conversely, using the kappa opioid receptor antagonist nalbuphine significantly attenuated scratching behavior, reduced PKCβ expression and p38 phosphorylation, and decreased spinal dorsal horn microglial activation, while PKCδ and KOR expression elevated. Spinal PKCβ silencing mitigated MIS and microglial activation. Still, knockdown of PKCδ reversed the inhibitory effect of nalbuphine on MIS and microglial activation, indicating that PKCδ is indispensable for the antipruritic effects of nalbuphine. In contrast, PKCβ is crucial for inducing microglial activation in MIS in male mice. Our findings show a distinct itch cascade of morphine, PKCβ/p38MAPK, and microglial activation, but an anti-MIS pathway of nalbuphine, PKCδ/KOR, and neuron activation.