Reactive oxygen species (ROS) play a vital role in reducing the viability of invading pathogens during plant-pathogen interactions. To understand how oxidative stress caused by ROS reduces cell viability, it is important to identify the proteins affected by ROS. In the present study, we investigated the changes in the expression of proteins from the outer and inner membrane fractions in Pseudomonas syringae pv tomato DC3000 under oxidative stress through membrane subproteomics. A total number of 17 differentially expressed proteins from the outer and inner membrane fractions were identified, among which 11 proteins belong to transporters, such as porins and ABC transporters. Their abundance was all decreased under oxidative stress, indicating that transporters are likely to be affected by oxidative stress. The function of two identified transporters was further characterized by constructing their gene mutant and overexpression strains. We found that mutation of one transporter gene PSPTO_1720 rendered Pseudomonas more sensitive to oxidative stress, whereas overexpression of this gene made the strain more resistant. By comparison, the mutant and overexpression strains of another transporter gene PSPTO_2152 exhibited the same sensitivity to oxidative stress compared with the wild-type. Our data suggest that oxidative stress reduces the viability of bacterial cells by acting on special transporters.