Updated project metadata. Bacterial divisome is a group of proteins governing cell division. Out of the 15 critical proteins in Escherichia coli, the transmembrane protein complex FtsQBL plays an essential role in regulating the action. Although extensive efforts have been made, there is limited information regarding the interaction within this three-member complex. Here, HDX-MS was used to study the dynamic change of the full-length protein FtsQ, FtsB, and FtsL. Direct experimental evidence shows that FtsB and FtsL form a stable complex by the transmembrane and periplasmic regions. After the complex formation, the structural feature in FtsB properly becomes more defined. This transition may also bring the constriction control domains (CCDs) close to the one in FtsL. Furthermore, with the helical interaction between two proteins, the CCDs have a more determined location relative to the membrane surface. On the other hand, the FtsQ in the FtsQBL complex showed rigidity enhancement in two regions. The interaction with FtsB would fix a significant portion of the FtsQ β-domains. Interestingly, the formation of the FtsQBL complex also stiffens the sequence connecting the two domains in FtsQ. Overall, this study provides important experimental evidence on the conformational dynamic change of the FtsQ, FtsB, and FtsL upon the complex formation and insights into the FtsQBL function in the divisome.