Abstract Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-hydroxycholesterol (25HC), a product of activity of cholesterol-25-hydroxylase (CH25H) upon cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against SARS-CoV-2. However, 25HC can also amplify inflammation and tissue injury and be converted by CYP7B1 to 7α,25HC, a lipid with chemoattractant activity via the G protein-coupled receptor, EBI2/GPR183. Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that while 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 mouse model in vivo . 25HC treatment also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma pro-inflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points, but no change in weight loss. Consistent with these findings, although Ch25h was upregulated in the lungs of SARS-CoV-2-infected WT mice, lung viral titers and weight loss in Ch25h −/– and Gpr183 −/– mice infected with the beta variant were similar to control animals. Taken together, endogenous 25-hydroxycholesterols do not significantly regulate early SARS-CoV-2 replication or pathogenesis and supplemental 25HC may have pro-injury rather than therapeutic effects in SARS-CoV-2 pneumonia.