We have developed and applied single live cell imaging for real-time monitoring of resistance kinetics of Pseudomonas aeruginosa. Real-time images of live cells in the presence of a particular substrate (EtBr) provided the first direct insights of resistance mechanism with both spatial and temporal information and showed that the substrate appeared to be accumulated in cytoplasmic space, but not periplasmic space. Three mutants of P. aeruginosa, PAO4290 (a wild-type expression level of MexAB-OprM), TNP030#1 (nalB-1, MexAB-OprM over expression mutant), and TNP076 (ΔABM, MexAB-OprM deficient mutant), were used to investigate the roles of these three membrane proteins (MexAB-OprM) in the resistance mechanism. Ethidium bromide (EtBr) was chosen as a fluorescence probe for spectroscopic measurement of bulk cell solution and single cell imaging of bulk cells. Bulk measurement indicated, among three mutants, that nalB-1 accumulated the least EtBr and showed the highest resistance to EtBr, whereas ΔABM accumulated the most EtBr and showed the lowest resistance to EtBr. This result demonstrated the MexAB-OprM proteins played the roles in resistance mechanism by extruding EtBr out of cells. Unlike the bulk measurement, imaging and analysis of bulk cells at single cell resolution demonstrated individual cell had its distinguished resistance kinetics and offered the direct observation of the regulation of influx and efflux of EtBr with both spatial and temporal resolution. Unlike fluorescent staining assays, live cell imaging provided the real-time kinetic information of transformation of membrane permeability and efflux pump machinery of three mutants. This research constitutes the first direct imaging of resistance mechanism of live bacterial cells at single cell resolution and opens up the new possibility of advancing the understanding of bacteria resistance mechanism.