The distribution of phospholipids across the inner membrane (IM) of Gram-negative bacteria is unknown. We demonstrate that the IMs of Escherichia coli and Yersinia pseudotuberculosis are asymmetric, with a 75%/25% (cytoplasmic/periplasmic leaflet) distribution of phosphatidylethanolamine (PE) in rod-shaped cells and an opposite distribution in E. coli filamentous cells. In initially filamentous PE-lacking E. coli cells, nascent PE appears first in the periplasmic leaflet. As the total PE content increases from nearly zero to 75%, cells progressively adopt a rod shape and PE appears in the cytoplasmic leaflet of the IM. The redistribution of PE influences the distribution of the other lipids between the leaflets. This correlates with the tendency of PE and cardiolipin to regulate antagonistically lipid order of the bilayer. The results suggest that PE asymmetry is metabolically controlled to balance temporally the net rates of synthesis and translocation, satisfy envelope growth capacity, and adjust bilayer chemical and physical properties.