Abstract: Finding the efficient antibiotic adjuvants and studying their synergistic bactericidal mechanism is one of the effective strategies to deal with the problem of bacterial resistance. In order to explore the mechanism of D-trehalose enhancing the killing effect of Escherichia coli by the aminoglycoside antibiotics, by taking Escherichia coli BW25113 as the research object, the contents of ATP, NADH and proton motive force (PMF) were measured before and after the treatment of D-trehalose combined with the aminoglycoside antibiotics. The changes of energy metabolism and membrane potential in bacteria were analyzed, and the effect on the uptake of antibiotics was detected by using the inhibition zone experiment. The results showed that: after the double treatment with D-trehalose combined with tobramycin or gentamycin, D-trehalose could increase the synthesis of ATP, increase the level of NADH, and promote the uptake of antibiotics by increasing the PMF level of Escherichia coli, thus significantly enhancing the bactericidal effect of antibiotics. The results of this study provided a basis for the development of D-trehalose as a new-type aminoglycoside antibiotic adjuvant.