The mycotoxin deoxynivalenol (DON) has a high global prevalence in grain-based products. Biomarkers of exposure are detectable in most humans and farm animals. Considering the acute emetic and chronic anorexigenic toxicity of DON, maximum levels for food and feed have been implemented by food authorities. The tolerable daily intake (TDI) is 1 µg/kg body weight (bw)/day for the sum of DON and its main derivatives, which was based on the no-observed adverse-effect level (NOAEL) of 100 µg DON/kg bw/day for anorexic effects in rodents. Chronic exposure to a low-DON dose can, however, also cause inflammation and imbalanced neurotransmitter levels. In the present study, we therefore investigated the impact of a 2-week exposure at the NOAEL in mice by performing behavioural experiments, monitoring brain activation by c-Fos expression, and analysing changes in the metabolomes of brain and serum. We found that DON affected neuronal activity and innate behaviour in both male and female mice. Metabolite profiles were differentiable between control and treated mice. The behavioural changes evidenced at NOAEL reduce the safety margin to the established TDI and may be indicative of a risk for human health.