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Pulsed electric field (PEF) is an up-to-date non-thermal processing technology with a wide range of applications in the food industry. The inactivation effect of PEF on <i>Escherichia coli</i> was different under different conditions. The <i>E. coli</i> inactivated number was 1.13 ± 0.01 lg CFU/mL when PEF was treated for 60 min and treated with 0.24 kV/cm. The treatment times were found to be positively correlated with the inactivation effect of PEF, and the number of <i>E. coli</i> was reduced by 3.09 ± 0.01 lg CFU/mL after 100 min of treatment. The inactivation assays showed that <i>E. coli</i> was inactivated at electrical intensity (0.24 kV/cm) within 100 min, providing an effective inactivating outcome for Gram-negative bacteria. The purpose of this work was to investigate the cellular level (morphological destruction, intracellular macromolecule damage, intracellular enzyme inactivation) as well as the molecular level via transcriptome analysis. Field Emission Scanning Electron Microscopy (TFESEM) and Transmission Electron Microscope (TEM) results demonstrated that cell permeability was disrupted after PEF treatment. Entocytes, including proteins and DNA, were markedly reduced after PEF treatment. In addition, the activities of Pyruvate Kinase (PK), Succinate Dehydrogenase (SDH), and Adenosine Triphosphatase (ATPase) were inhibited remarkably for PEF-treated samples. Transcriptome sequencing results showed that differentially expressed genes (DEGs) related to the biosynthesis of the cell membrane, DNA replication and repair, energy metabolism, and mobility were significantly affected. In conclusion, membrane damage, energy metabolism disruption, and other pathways are important mechanisms of PEF’s inhibitory effect on <i>E. coli</i>.