Bacterial mechanosensitive channels are some of the largest pores in nature. In particular, MscL, with a pore diameter >25 Å, allows passage of large organic ions and small proteins. Functional MscL reconstitution into lipids has been proposed for applications in vesicular-based drug release. Here we show that these channels can be functionally expressed in mammalian cells to afford rapid controlled uptake of membrane-impermeable molecules. We first demonstrate that MscL gating in response to increased membrane tension is preserved in mammalian cell membranes. Molecular delivery is controlled by adopting an established method of MscL charge-induced activation. We then determine pore size limitations using fluorescently labelled model cargoes. Finally, we activate MscL to introduce the cell-impermeable bi-cyclic peptide phalloidin, a specific marker for actin filaments, into cells. We propose that MscL will be a useful tool for gated and controlled delivery of bioactive molecules into cells.