Among food proteins, alpha-lactalbumin (LAC) has the highest ratio of tryptophan (Trp) over its competitor amino acids. Consequently, contrary to casein (CAS), LAC ingestion increases Trp access to the brain leading to enhanced serotonin (5-HT) synthesis. As an index of serotonergic activity, we assessed extracellular 5-HT in response to LAC ingestion, using microdialysis, and performed behavioural tests in rats in order to characterise the suggested improvements of mood observed in humans after ingestion of this protein. Rats were fed with diets enriched either in LAC or CAS as control, acutely (30 min meals) or chronically (3 and 6 days). A 30 min LAC meal significantly increased 5-HT release in the medial hypothalamus. This effect disappeared after 3 and 6 days of diet. The basal premeal 5-HT levels were increasingly enhanced by the LAC diet. Compared to a CAS meal, LAC increased the percentage of time spent on the open arms of the elevated plus maze and the number of visits to the centre of the open field, suggesting an anxiolytic-like effect. A single LAC meal decreased sucrose consumption, while 3 or 6 days diets enhanced it, reflecting an appetitive and/or rewarding action. In conclusion, LAC ingestion induces anxiolytic-like and rewarding effects possibly related to serotonergic activation. Shifting transiently, the commonly consumed CAS-enriched to LAC-enriched diets may induce beneficial effects on mood.