[Objective] The important foodborne bacterial pathogen Listeria monocytogenes uses the glutaredoxin (Grx) system to defend the oxidative stress during environmental adaption. Here, we explore biological characteristics of the glutathione reductase (GR) during bacterial infection. [Methods] We constructed the gr deletion and complementation strains to compare the abilities in bacterial growth, motility, oxidative tolerance and cellular infection. Besides, the gr promoter-based fluorescent report system (FRS) was genetically obtained to further study regulation of gr by Grx. [Results] Our data show that deletion of gr did not affect bacterial growth but enhanced the capability of swarming. Additionally, lack of gr significantly increased oxidative tolerance of bacteria under Cu²⁺ and Cd²⁺ stress, as well as increased efficiencies of intracellular infection in Caco-2 and RAW264.7 cells. Importantly, transcription of gr was negatively regulated by Grx. [Conclusion] The glutathione reductase plays a critical role in bacterial flagella-based swarming motility and correlates a regulatory relationship with the Grx system. Also, GR plays a non-classical role in bacterial oxidative resistance and intracellular infection. This study will expand our understanding of the redox regulatory mechanisms exploited by intracellular bacteria during adaption outside the environment and inside the host, which provides novel strategies to prevent and control the foodborne diseases.