Regulators of G protein Signaling (RGS) proteins negatively modulate presynaptic μ-opioid receptor inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). Paradoxically， we find that G protein-coupled receptor (GPCR) activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels in the vlPAG is reduced in an agonist- and receptor-dependent manner in transgenic knock-in mice of either sex expressing mutant RGS-insensitive Gαo proteins. μ-Opioid receptor agonist activation of GIRK currents was reduced for DAMGO and fentanyl but not for [met]-enkephalin (ME) in the RGS-insensitive heterozygous (Het) mice compared to wildtype mice. The GABAB agonist baclofen-induced GIRK currents were also reduced in the Het mice. We confirmed the role of Gαo proteins in μ-opioid receptor and GABAB receptor signaling pathways in wildtype mice using myristoylated-peptide inhibitors of Gαo and Gαi The results using these inhibitors indicate that receptor activation of GIRK channels is dependent on preference of the agonist-stimulated receptor for Gαo versus Gαi. DAMGO and fentanyl-mediated GIRK currents were reduced in the presence of the Gαo inhibitor， but not the Gαi inhibitors. In contrast， the Gαo peptide inhibitor did not affect ME activation of GIRK currents， consistent with results in the Het mice， but the Gαi inhibitors significantly reduced ME-mediated GIRK currents. Finally， the reduction in GIRK activation in the Het mice plays a role in opioid and baclofen-mediated spinal， but not supraspinal antinociception. Thus， our studies indicate that RGS proteins have multiple mechanisms of modulating GPCR signaling that produce negative and positive regulation of signaling depending on the effector. RGS proteins positively modulate GPCR coupling to GIRK channels and this coupling is critical for opioid and baclofen-mediated spinal antinociception， whereas μ-opioid receptor-mediated supraspinal antinociception depends on presynaptic inhibition that is negatively regulated by RGS proteins. The identification of these opposite roles for RGS proteins has implications for signaling via other GPCRs.