TRPM3 proteins form Ca2+-permeable, non-selective cation channels that can be activated by diverse chemical compounds, notably the steroid pregnenolone sulfate, and by some physical stimuli, principally by heat. Accordingly, TRPM3 channels have been shown to play important roles in heat-induced nociception and in inflammatory thermal hyperalgesia (1, 2). Recently, we and others have shown that these channels are tightly regulated by Gαi-coupled receptors (3 – 5). Notably, in peripheral nociceptor neurons µ-opioid receptors have a strong inhibitory influence on TRPM3 channels. Hence, the peripheral activation of µ-opioid receptors reduces TRPM3-dependent pain in vivo. The signaling pathway from µ-opioid receptors to TRPM3 channels was shown to be dependent on Gβγ subunits (3 – 5). However, it is not clear, whether TRPM3 channels and Gβγ subunits interact directly, and if so, where on the respective proteins the interaction takes place. By screening naturally occurring isoforms of TRPM3 arising from alternative splicing, we discovered that a region of 10 amino acids, encoded by exon 17, is required for the inhibitory action of Gβγ subunits on TRPM3. Mutational analysis indicated that 5 of these 10 residues are particularly important for this functional interaction. Biochemically, we showed that Gβ proteins that co-immunoprecipitated with TRPM3 were much reduced for a TRPM3 isoform that lacks exon 17-encoded amino acids. Conversely, short peptides containing parts or all of the sequence encoded by exon 17 preferentially displayed binding to Gβγ proteins in vitro. Finally, X-ray analysis of Gβγ co-crystallized with a short peptide encoded by exon 17 showed that this peptide adopts an α-helical structure to interact with Gβ. The molecular interactions observed in the crystal structure involved mainly the same amino acids previously identified through mutagenesis as important for the functional Gβγ-mediated inhibition of TRPM3. Conversely, structure-guided mutations of Gβ proteins demonstrated that residues of Gβ, which interacted with the exon 17-encoded peptide in the crystal structure, are also important for the functional inhibitory effects of Gβγ on TRPM3 in cellular assays. Together, these data provide evidence of considerable strength that Gβ directly interacts with TRPM3 proteins through amino acids of TRPM3 encoded by exon 17 and that this direct interaction is crucial for the inhibitory action of Gβγ on TRPM3. The interaction surface between Gβ and TRPM3 that we have identified might be druggable and thus presents a good opportunity to develop selective TRPM3 inhibitors for the treatment of TRPM3-dependent pain.