Abstract One of the hallmarks of critically ill COVID-19 patients infected with SARS-CoV-2 is exaggerated inflammatory response. Though macrophages mediate inflammatory responses and can produce pro-inflammatory cytokines to eliminate pathogens, infection with SARS-CoV-2 has been shown to cause immune dysfunction, leading to hyperinflammation in the lungs. To further understand the role of macrophages in hyperinflammatory responses during SARS-CoV-2 infection, we infected a THP-1 human derived macrophage cell line with SARS-CoV-2. Our results show that, though macrophages do not support viral replication, infection with SARS-CoV-2 still results in the upregulation of the mRNA of cytokines TNFα and CXCL10, which are markers of COVID-related hyperinflammation. In addition, we identified SARS-CoV-2 Spike protein S1 subunit as one viral factor involved in the upregulation of cytokines in macrophages. We show that glycosylated, soluble S1 protein can upregulate TNFα and CXCL10 mRNAs, as well as the secretion of TNFα, in macrophages in the absence of virus infection. Therefore, macrophage activation by the S1 subunit and SARS-CoV-2 infection may contribute to the hyperinflammation in the lungs seen in critically ill patients through the upregulation of proinflammatory cytokines such as TNFα and CXCL10. Supported by NIH grant (R01 AI083387), Washington Research Foundation, and NIH NIGMS Predoctoral Biotechnology Training Grant 5T32GM008336