Antibiotic-resistant Vibrio alginolyticus poses a big challenge to human health and food safety. It is urgently needed to understand the mechanisms underlying antibiotic resistance to develop effective approaches for the control. Here we explored the metabolic difference between gentamicin-resistant V. alginolyticus (VA-RGEN ) and gentamicin-sensitive V. alginolyticus (VA-S), and found that the reactive oxygen species (ROS) generation was altered. Compared with VA-S, the ROS content in VA-RGEN was reduced due to the decreased generation and increased breakdown of ROS. The decreased production of ROS was attributed to the decreased central carbon metabolism, which is associated with the resistance to gentamicin. As such a mechanism, we exogenously administrated VA-RGEN with the glucose that activated the central carbon metabolism and promoted the generation of ROS, but decreased the breakdown of ROS in VA-RGEN . The gentamicin-mediated killing was increased with the elevation of the ROS level by a synergistic effect between gentamicin and exogenous glucose. The synergistic effect was inhibited by thiourea, a scavenger of ROS. These results reveal a reduced ROS-mediated antibiotic resistance mechanism and its reversal by exogenous glucose.