β-Lactotensin (His-Ile-Arg-Leu) is a bioactive peptide derived from bovine milk β-lactoglobulin, acting as a natural agonist for neurotensin receptors. We found that β-lactotensin exhibited anxiolytic-like activity in an elevated plus-maze test after its intraperitoneal (i.p.) administration in mice. β-Lactotensin was also orally active. The anxiolytic-like activity of β-lactotensin after i.p. administration was blocked by levocabastine, an antagonist for the neurotensin NTS(2) receptor. β-Lactotensin had anxiolytic-like activity in wild-type but not Ntsr2-knockout mice. β-Lactotensin increased intracellular Ca(2+) flux in glial cells derived from wild-type mice but not Ntsr2 knockout mice. These results suggest that β-lactotensin acts as an NTS(2) receptor agonist having anxiolytic-like activity. The anxiolytic-like activity of β-lactotensin was also blocked by SCH23390 and SKF83566, antagonists for dopamine D(1) receptor, but not by raclopride, an antagonist for D(2) receptor. Taken together, β-lactotensin may exhibit anxiolytic-like activity via NTS(2) receptor followed by D(1) receptor.