Due to the unique bacterial killing ability through membrane permeabilization and content leakage, antimicrobial peptides (AMPs) have been regarded as promising candidates against the severe threats of drug-resistant bacteria and even superbugs to public health. However, investigations of the mechanism underlying their membrane permeabilization like poration are still on the way. Here, from the perspective of molecular motion kinetics, we studied the interactions between melittin, as one of the most representative AMPs, and a bi-component lipid membrane based on the combination of single-molecule tracking and molecular dynamics simulations. Our results reveal that, the mobility of some lipids in membrane, in comparison of the other most molecules, is significantly decreased by the surface adsorption and transmembrane insertion of melittin. Moreover, melittin tends to work at the boundary region between phase domains, disturb and blur the phase separation behavior and consequently lower the confinement of phase boundary on lipid motions. This work demonstrates the correlation between membrane activity of melittin and the motion kinetics of lipids as well as phase behavior of the membrane. These results would be helpful not only for understanding the molecular mechanism of AMPs from a new perspective but also for the development of new antibacterial agents with improved performance.