Abstract: The SARS-CoV-2 spike protein engages the human ACE2 receptor through a conformationally-dynamic receptor-binding domain (RBD) to initiate viral entry. Evidence supports a pathway for SARS-CoV-2 to enter the host through low-pH endosomes. To understand how Spike conformation is affected by a low-pH environment, cryo-electron microscopy structures at serological and endosomal pH were determined and revealed that Spike adopts a conformation with each RBD in the down position at a low pH. This was mediated through structural rearrangements in a newly-discovered pH-dependent refolding region. These results may also reveal new insights into how SARS-CoV-2 evades immune recognition as binding between Spike and a known anti-SARS-CoV-2 Spike antibody was diminished.