AbstractOne of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site‐directed spin labeling (SDSL‐EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin‐labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site‐specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time‐resolved dynamics of NarJ in cellular context.