Individual data used to generate Figs 2D and S7B.

The human glucose-6-phosphate transporter (G6PT/SLC37A4) mediates the translocation of glucose-6-phosphate (G6P) from the cytoplasm into the endoplasmic reticulum, a process essential for glucose production and the maintenance of blood glucose homeostasis between meals. Dysfunction of G6PT causes glycogen storage disease type Ib (GSD-Ib), a severe metabolic disorder characterized by hypoglycemia, hepatomegaly, and neutropenia. Despite its physiological and clinical significance, the structural basis of G6P recognition and the molecular mechanisms underlying GSD-Ib have remained elusive. Here, we present cryo-electron microscopy structures of human G6PT, revealing a monomer in an outward-open state at 3.1 Å and a homodimeric assembly in a face-to-face topology at 3.3 Å. By combining computational modeling of the G6P–G6PT complexes with functional characterization, we have uncovered the key molecular elements that govern the alternating-access mechanism: an electropositive substrate-binding pocket tailored for phosphorylated sugars; conserved aromatic residues that seal the cytosolic gate; and a dynamic inter-domain salt bridge that regulates the conformational transition. Our work provides fundamental insights into the transport cycle of the organophosphate:phosphate antiporter (OPA) family, offers a framework for interpreting GSD-Ib pathology at the molecular level, and establishes a foundation for advancing the mechanistic understanding of the human SLC37 family.