The shape of bacteria is maintained by the cell wall. The main component of the cell wall is peptidoglycan (PG) that is synthesized by penicillin binding proteins (PBPs). The correct positioning of PBPs is essential for the maintenance of cell shape. In the literature, two different models for localization of PBPs have been proposed – localization through interaction with a cytoskeletal structure or localization through the presence of substrate. Here, we show that the localization of PBPs critical for the rod shape of Bacillus subtilis is altered when the substrate LipidII is delocalized by treatment of the cells with nisin. Alteration of this localization is only seen in a LipidII-dependent manner and is not influenced by dissipation of the membrane potential, a secondary effect of nisin treatment. Our results strongly suggest that the localization of PG synthesis at the periphery of the cell is substrate-driven, even in bacteria that contain actin-like MreB cytoskeletal structures.