Rhomboid proteases are integral membrane serine proteases that catalyze peptide bond hydrolysis in biological membranes. Little is currently known about the interaction of enzyme and substrate. Coarse-grained molecular dynamics simulations in hydrated lipid bilayers are employed herein to study the interaction of the E. coli rhomboid protease GlpG (ecGlpG) with the transmembrane domain (TMD) of the substrate Spitz. Spitz does not associate with ecGlpG exclusively at the putative substrate gate near TMD 5. Instead, there are six prominent and stable interaction sites, including one between TMDs 1 and 3, with the closest enzyme-substrate proximity occurring at the ends of helical TMDs or in loops. Bilayer thinning is observed proximal to ecGlpG, but there is no evidence of additional thinning of the bilayer upon interaction with substrate. We suggest that the initial interaction between enzyme and substrate, or substrate capture event, is not limited to a single site on the enzyme, and may be driven by juxtamembrane electrostatic interactions. The findings are of additional interest because catalytically inactive rhomboids (iRhoms) are now known to interact with the substrates of their catalytically active counterparts and to antagonize the enzyme-driven pathways.