Inter-helix hydrogen bonding involving asparagine (Asn, N), glutamine (Gin, Q), aspartic acid (Asp, D) or glutamic acid (Glu, E) can drive efficient di- or trimerization of transmembrane helices in detergent micelles and lipid bilayers. Likewise, Asn-Asn and Asp-Asp pairs can promote the formation of helical hairpins during translocon-mediated membrane protein assembly in the endoplasmic reticulum. By in vitro translation of model integral membrane protein constructs in the presence of rough microsomes, we show that Asn- or Asp-mediated interactions with a neighbouring transmembrane helix can enhance the membrane insertion efficiency of a marginally hydrophobic transmembrane segment. Our observations suggest that inter-helix hydrogen bonds can form during Sec61 translocon-assisted insertion and thus could be important for membrane protein assembly.