Background: Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. No genome-wide study focusing on the changes of gene expression by aminoglycosides in E.coli has been reported. Here, we report transcriptome-profiling analysis of E.coli with or without Kanamycin B to elucidate the understanding of non-antibiotic cellular functions. Results: The differentially expressed genes (DEGs) at two given concentrations of Kanamycin B were identified. The results indicated that Kanamycin B does not affect the expression of the majority of the genes. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. Conclusions: Kanamycin B treatment causes diverse changes in the transcriptional profile of E. coli JM109, that are not directly associated with the antibiotic activity of Kanamycin B.