Enzyme IIA^Glc (EIIA^Glc) of the phosphoenolpyruvate phosphotransferase system for the uptake of glucose in Escherichia coli and Salmonella inhibits the maltose ATP-binding cassette transporter (MalE-FGK₂) by interaction with the nucleotide-binding and -hydrolyzing subunit MalK, a process termed inducer exclusion. We have investigated binding of EIIA^Glc to the MalK dimer by cysteine cross-linking in proteoliposomes. The results prove that the binding site I of EIIA^Glc is contacting the N-terminal subdomain of MalK while the binding site II is relatively close to the C-terminal (regulatory) subdomain, in agreement with a crystal structure [Chen, S., Oldham, M. L., Davidson, A. L., and Chen, J. (2013) Nature499, 364−368]. Moreover, EIIA^Glc was found to bind to the MalK dimer regardless of its conformational state. Deletion of the amphipathic N-terminal peptide of EIIA^Glc, which is required for inhibition, reduced formation of cross-linked products. Using a spin-labeled transporter variant and EPR spectroscopy, we demonstrate that EIIA^Glc arrests the transport cycle by inhibiting the ATP-dependent closure of the MalK dimer.