Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors may also play major roles in viral infection. Here we report that the stress-inducible molecular chaperone GRP78 can form a complex with the SARS-CoV-2 Spike protein and ACE2 intracellularly and on the cell surface, and that the substrate binding domain of GRP78 is critical for this function. Knock-down of GRP78 by siRNA dramatically reduced cell surface ACE2 expression. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159), selected for its ability to cause GRP78 endocytosis and its safe clinical profile in preclinical models, reduces cell surface ACE2 expression, SARS-CoV-2 Spike-driven viral entry, and significantly inhibits SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78.