Pyrene is a pollutant in the environment and affects the health of living organisms. It is important to understand microbial-mediated pyrene resistance and the related molecular mechanisms due to its toxicity and biodegradability. Due to the unclear response mechanisms of bacteria to PAHs, this study detected the transcriptional changes in Escherichia coli under different pyrene concentrations using transcriptome sequencing technology. Global transcriptome analysis showed that the number of differentially expressed genes (DEGs) in multiple metabolic pathways increased with increasing concentrations of pyrene. In addition, the effects and toxicity of pyrene on Escherichia coli mainly included the up-regulation and inhibition of genes related to carbohydrate metabolism, membrane transport, sulfate reduction, various oxidoreductases, and multidrug efflux pumps. Moreover, we also constructed an association network between significantly differentially expressed sRNAs and key genes and determined the regulatory relationship and key genes of Escherichia coli under pyrene stress. Our study utilized pyrene as an exogenous stress substance to investigate the possible pathways of the bacterial stress response. In addition, this study provides a reference for other related research and serves as a foundation for future research.