We report the design of novel biomimetic membrane model and its use to characterize in vitro the translocation process of a bacterial toxin, the adenylate cyclase (CyaA) from Bordetella pertussis. A tethered lipid bilayer was assembled over a gold surface derivatized with calmodulin (CaM) on top of an SPR (Surface Plasmon Resonance) biosensor. The assembled bilayer formed a continuous and protein-impermeable boundary fully insulating the underlying CaM (trans side) from the above medium (cis side). SPR spectroscopy was used to monitor toxin binding to the membrane, while activation of the catalytic activity of CyaA by the tethered CaM was used as a reporter of its translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent upon the presence of calcium, and upon application of a negative trans-membrane potential, in good agreement with prior studies done on eukaryotic cells. These results demonstrate that CyaA does not require any specific eukaryotic components to translocate across membrane and suggest that the translocation mechanism involves an electrophoretic transport of the toxin catalytic domain driven by the electrical field across the membrane. To our knowledge, this work constitutes the first in vitro demonstration of toxin translocation across a tethered lipid bilayer. This biomimetic assembly opens new opportunities to explore the molecular mechanisms of protein translocation across biological membranes.