The bacterial mechanosensitive channel of small conductance (MscS) plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. The functional characteristics of MscS have been extensively studied using liposomal reconstitution. This is a widely used experimental paradigm and is particularly important for mechanosensitive channels as channel activity can be probed free from cytoskeletal influence. A perpetual issue encountered using this paradigm is unknown channel orientation. Here we examine the orientation of MscS in liposomes formed using 2 ion channel reconstitution methods employing the powerful combination of patch clamp electrophysiology, confocal microscopy, and continuum mechanics simulation. Using the previously determined electrophysiological and pharmacological properties of MscS, we were able to determine that in liposomes, independent of lipid composition, MscS adopts the same orientation seen in native membranes. These results strongly support the idea that these specific methods result in uniform incorporation of membrane ion channels and caution against making assumptions about mechanosensitive channel orientation using the stimulus type alone.