ABSTRACTCoxiella burnetiiis an intracellular bacterium that causes the human disease Q fever.C. burnetiiforms a large, acidicCoxiella-containing vacuole (CCV) and uses a type 4B secretion system to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting thatC. burnetiiregulation of lipid transport and metabolism is critical for successful infection. The mammalian lipid transport protein ORP1L (oxysterol binding protein-like protein 1 Long) localizes to the CCV membrane and mediates CCV-endoplasmic reticulum (ER) membrane contact sites. ORP1L functions in lipid sensing and transport, including cholesterol efflux from late endosomes and lysosomes (LELs), and the ER. Its sister isoform, ORP1S (oxysterol binding protein-like protein 1 Short) also binds cholesterol but has cytoplasmic and nuclear localization. In ORP1-null cells, we found that CCVs were smaller than in wild-type cells, highlighting the importance of ORP1 in CCV development. This effect was consistent between HeLa cells and murine alveolar macrophages (MH-S cells). CCVs in ORP1-null cells had higher cholesterol content than CCVs in wild-type cells at 4 days of infection, suggesting ORP1 functions in cholesterol efflux from the CCV. While the absence of ORP1 led to aC. burnetiigrowth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrated thatC. burnetiiuses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol.IMPORTANCECoxiella burnetiiis an emerging zoonotic pathogen and bioterrorism threat. No licensed vaccine exists in the United States, and the chronic form of the disease is difficult to treat and potentially lethal. Postinfectious sequelae ofC. burnetiiinfection, including debilitating fatigue, place a significant burden on individuals and communities recovering from an outbreak.C. burnetiimust manipulate host cell processes in order to promote infection. Our results establish a link between host cell lipid transport processes andC. burnetii’s avoidance of cholesterol toxicity during infection of alveolar macrophages. Elucidating the mechanisms behind bacterial manipulation of the host will yield insight for new strategies to combat this intracellular pathogen.