UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin ? (T?) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like ?-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated G? peptides. The structure of co-crystals of UNC119 with an acylated T? N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound T?-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-T?-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a G? subunit cofactor essential for G protein trafficking in sensory cilia.