In both clinical and preclinical scenarios, 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG) is the radiotracer most widely used to study brain glucose metabolism with positron emission tomography (PET). In clinical practice, there is a worldwide standardized protocol for preparing patients for [18F]FDG-PET studies, which specifies the room lighting. However, this standard is typically not observed in the preclinical field, although it is well known that animal handling affects the biodistribution of [18F]FDG. The present study aimed to evaluate the effect of ambient lighting on brain [18F]FDG uptake in mice. Two [18F]FDG-PET studies were performed on each animal, one in light and one in dark conditions. Thermal video recordings were acquired to analyse animal motor activity in both conditions. [18F]FDG-PET images were analysed with the Statistical Parametric Mapping method. The results showed that [18F]FDG uptake is higher in darkness than in light condition in mouse nucleus accumbens, hippocampus, midbrain, hindbrain, and cerebellum. The SPM analysis also showed an interaction between the illumination condition and the sex of the animal. Mouse activity was significantly different (p = 0.01) between light conditions (632 ± 215 s of movement) and dark conditions (989 ± 200 s), without significant effect of sex (p = 0.416). We concluded that room illumination conditions during [18F]FDG uptake in mice affected the brain [18F]FDG biodistribution. Therefore, we highlight the importance to control this factor to ensure more reliable and reproducible mouse brain [18F]FDG-PET results.