We report the design of novel biomimetic membrane model and its use to characterize in vitro the translocation process of bacterial toxin, the adenylate cyclase (CyaA) from Bordetella pertussis. The membrane was assembled over a calmodulin (CaM) layer and exhibits the fundamental characteristics of a biological membrane separating two cis and trans compartments. SPR was used to monitor the membrane interaction of the CyaA toxin, while the activation of the catalytic activity of CyaA by the tethered CaM was used as a probe 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 eukaryotic components to translocate across a membrane, and suggest that CyaA is electrophoretically transported across the bilayer by the transmembrane electrical field. To our knowledge, this work constitutes the first in vitro demonstration of protein translocation across a tethered lipid bilayer. This biomimetic assembly opens new opportunities to explore the molecular mechanisms of protein translocation across biological membranes.