Auriculo-condylar syndrome (ACS), a rare condition that impairs craniofacial development, is caused by mutations in a G protein–coupled receptor (GPCR) signaling pathway. In mice, disruption of signaling by the endothelin type A receptor (ET_AR), which is mediated by the G protein (heterotrimeric guanine nucleotide–binding protein) subunit Gα_q/11 and subsequently phospholipase C (PLC), impairs neural crest cell differentiation that is required for normal craniofacial development. Some ACS patients have mutations in GNAI3, which encodes Gα_i3, but it is unknown whether this G protein has a role within the ET_AR pathway. We used a Xenopus model of vertebrate development, in vitro biochemistry, and biosensors of G protein activity in mammalian cells to systematically characterize the phenotype and function of all known ACS-associated Gα_i3 mutants. We found that ACS-associated mutations in GNAI3 produce dominant-negative Gα_i3 mutant proteins that couple to ET_AR but cannot bind and hydrolyze guanosine triphosphate, resulting in the prevention of endothelin-mediated activation of Gα_q/11 and PLC. Thus, ACS is caused by functionally dominant-negative mutations in a heterotrimeric G protein subunit.