beta-Lactamase inhibitory protein (BLIP) can effectively inactivate class A beta-lactamases, but with very different degrees of potency. Understanding the different roles of BLIP in class A beta-lactamases inhibition can provide insights for inhibitor design. However, this problem was poorly solved on the basis of the static structures obtained by X-ray crystallography. In this work, ion mobility mass spectrometry, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation revealed the conformational dynamics of three class A beta-lactamases with varying inhibition efficiencies by BLIP. A more extended conformation of PC1 was shown compared to those of TEM1 and SHV1. Localized dynamics differed in several important loop regions, that is, the protruding loop, H10 loop, Omega loop, and SDN loop. Upon binding with BLIP, these loops cooperatively rearranged to enhance the binding interface and to inactivate the catalytic sites. In particular, unfavorable changes in conformational dynamics were found in the protruding loop of SHV1 and PC1, showing less effective binding. Intriguingly, the single mutation on BLIP could compensate for the unfavored changes in this region, and thus exhibit enhanced inhibition toward SHV1 and PC1. Additionally, the H10 region was revealed as an important allosteric site that could modulate the inhibition of class A beta-lactamases. It was suggested that the rigid protruding loop and flexible H10 region might be determinants for the effective inhibition of TEM1. Our findings provided unique and explicit insights into the conformational dynamics of beta-lactamases and their bindings with BLIP. This work can be extended to other beta-lactamases of interest and inspire the design of novel inhibitors.