Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the 15N isotope effect on Escherichia coli cultures that were grown in either unlabeled (14N) or 15N-labeled media by LC-ESI-MS/MS-based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between 14N and 15N-labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between 14N and 15N-labeled bacteria. Our data demonstrate for the first time that the introduction of the 15N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.