Bacterial cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding to the cytoplasmic minidomains of peptidoglycan synthases to ensure their correct subcellular localisation. A combination of structural biology, biochemistry and bacterial genetics has revealed features critical for the interaction of GpsB from different bacteria with peptidoglycan synthases. We have used these data to predict and confirm novel partners of GpsB, illuminating the role of this key regulator of peptidoglycan synthesis. Given the importance of GpsB in pathogenic bacteria, this study presents a start point for the design of much needed new antibiotics.