Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the μ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCβ3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization. Display omitted ► Unique isoforms of the μ-opioid receptor (MOR) separate itch from analgesia ► Heterodimers of MOR1D and gastrin-releasing peptide receptor (GRPR) form ► Molecular basis for opioid-induced itch relies on MOR1D activation of GRPR ► PLCβ3 and IP3R3 signal downstream of MOR1D-GRPR to trigger itch response Itching is an unwanted side effect of opioid-mediated pain relief. This study reveals how distinct populations of spinal cord neurons differentially respond to morphine, producing either itch or analgesia.