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Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria species produce sterols, but their function in the bacterial domain remains unknown. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. Subsequent studies demonstrated that C-4 methylated sterols synthesized in the cytosol are localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. While proteins involved in eukaryotic sterol transport have been identified and characterized, their counterparts in bacteria remain enigmatic. In this study, we used bioinformatics, ligand binding analysis, and structural approaches to identify three novel bacterial sterol transporters from Methylococcus capsulatus. These proteins reside in the inner membrane, periplasm, and outer member of the bacterium, thereby working as a conduit to move modified sterols to the outer membrane. We report their remarkable specificity for recognizing 4-methylsterols, reveal the structural bases for substrate binding, and describe their structural divergence from eukaryotic sterol transporters. These findings provide unequivocal evidence for a distinct sterol transport system within the bacterial domain, which provide insights into our understanding of bacterial sterols and their divergence from similar lipids in higher-order organisms.SignificanceSterol lipids, such as cholesterol, were once thought to be unique to eukaryotes. However, a few bacterial species also produce sterols, albeit with structural modifications not observed in eukaryotes. While little is known about the function of bacterial sterols, it is known that sterols in certain bacteria are localized to the outer membrane. However, how bacteria transport these lipids from the cytoplasm to the outer membrane remains unknown. Here, we identify three novel bacterial sterol transporter proteins in the bacterium Methylococcus capsulatus that bind sterols methylated at the C-4 position. Through structural characterization of these proteins, we reveal insights into the mechanisms of sterol transport in bacteria with implications for understanding how bacterial cells interact with sterol lipids more broadly.