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Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion. • Cryo-EM structures and native-MS of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 • The GET insertase forms a heterotetramer upon Get3 and interfacial lipid binding • A membrane-embedded hydrophilic groove is conserved with YidC and the EMC • Get2/CAML helix α3′ interacts with Get3/TRC40 to promote tail-anchored insertion McDowell et al. describe structures of the GET insertase bound to the Get3 cytoplasmic chaperone. They reveal that membrane insertion of tail-anchored proteins by Get1/Get2 homologs requires the concerted action of a lipid-stabilized heterotetramer, a conserved cytoplasmic helix α3′, and a membrane-embedded hydrophilic groove. [ABSTRACT FROM AUTHOR]