Bioluminescence is a chemical process that is widespread in Nature. In many cases, it stems from the oxidation of a luciferin molecule that is catalyzed by an enzyme, dubbed luciferase, resulting in the emission of a visible light photon. NanoLuc is a small and bright luciferase derived from the catalytic domain of a shrimp luciferase that uses coelenterazine as substrate. The enzyme has been shown to exhibit a high tolerance for substrate modification, allowing the development of optimized substrates and red-shifted substrate analogues. Despite its paramount interest in the toolbox of cell biologists, its development has been hindered by the lack of structural information. While the recent determination of the crystallographic structure of the apo-enzyme has unveiled its structural organization, it has raised the question of the accommodation of substrates within the active-site. Our work provide a comparison of NanoLuc structures obtained in three different space groups, highlighting the dynamics of loops capping the active-site. In addition, we have determined the first crystallographic structure of NanoLuc in complex with an inhibitor of the luciferase activity. This structure gives a glimpse of the loop rearrangements and key residues that are necessary for substrate accommodation, shedding light for further engineering efforts.