The α subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) in G protein-coupled receptor (GPCR) pathways operate as gate switches by cycling through inactive GDP-bound and active GTP-bound states. Three flexible regions (switches I to III) of Gα subunits undergo the most substantial conformational rearrangements and determine interactions with receptors, signaling regulators, and effectors. Here, we describe three patients with severe pediatric encephalopathy harboring c.723+1G>A or c.723+2T>A variants in the splicing donor site of intron 6 of GNAO1 , the gene encoding the major neuronal G protein α subunit Gα_o . These substitutions destroyed the conserved GU sequence of the pre-mRNA and rendered the donor site unrecognizable, prompting cryptic splice site engagement and production of the dominant pathogenic Gα_o [V234_T241del] variant, which lacked switch III (amino acid residues 232 to 243 in Gα_o ). Structural, biochemical, and cellular analyses characterized V234_T241del as a strong neomorphic variant that was severely deficient in guanine nucleotide handling and cellular interactions and sensitive to zinc salts, an emerging targeted treatment for a GNAO1 encephalopathy subgroup. Our studies provide pathological and molecular insights into the function and effect of removal of an entire switch region in 1 of the 16 human G protein α subunits that underlie multiple physiological processes and diverse pathologies.