To explore whether oxidative stress caused by 100% CO 2 is an inhibitory mechanism against Shewanella putrefaciens, the oxidative stress reaction, antioxidant activity, and damage to the cell membrane, protein, and DNA of CO 2 -incubated S. putrefaciens at 4 °C were evaluated. Research demonstrated that CO 2 caused more severe reactive oxygen species (ROS) accumulation. Simultaneously, weaker•− -scavenging activity and decreased T-VOC and GSH content were also observed. The activities of antioxidant enzymes (SOD, POD, CAT, and GPX) continuously declined, which might be attributed to the CO 2 -mediated decrease in the pH value. Correspondingly, the cell membrane was damaged with hyperpolarization, increased permeability, and more severe lipid peroxidation. The expression of total and membrane protein decreased, and the synthesis and activity of extracellular protease were inhibited. DNA was also subjected to oxidative damage and expressed at a lower level. All results collaboratively confirmed that ROS excitation and inhibition of antioxidant activity were important inhibition mechanisms of CO 2 on S. putrefaciens.