Mechanosensitive channel of large conductance (MscL) is a nonselective channel found in the plasma membrane of E. coli. It serves as an emergency release valve that protects cells from lysis by allowing the efflux of osmolytes under acute osmotic downshock. Studies of the MscL in yeast models and reconstituted liposomes have suggested that the channel can directly sense biophysical changes in its membrane environment. In particular, the channel is found to be gated by membrane tension on the order of 7-13 dynes·cm−2 and can have lower activation threshold with increased hydrophobic mismatch between the channel and lipid bilayer. Although MscL is the best characterized mechanosensitive channel, its mechanical gating and potential modes of activation when expressed in mammalian cells have not been fully investigated. To better understand the channel gating properties in mammalian cells, we employed different methods of mechanical stress application to human retinal pigment epithelial (RPE) cells expressing MscL WT and G22S, a mutant MscL with lower activation threshold. RPE cells were made to express MscL WT and G22S via infection of tetracycline regulated adenovirus vectors. We found that MscL WT and G22S were activated to allow influx of a fluorescent dye that increased with increasing osmotic downshock. Acoustic tweezing, a method akin to magnetic tweezing that applies acoustic pressure to displace microbubbles, was used to exert mechanical stress to the cell membrane. Microbubbles were functionalized to attach either to integrins or to transmembrane transferrin receptors. We found that MscL can be activated and the threshold of activation depends on the coupling of force transduction. Our results suggest that MscL retains its mechanical gating in mammalian cells and support the potential for MscL to be utilized as a mechanosensor in mammalian cells.