L-Ornithine has recently received considerable attention as a functional amino acid in the fields of nutrition science and health science due to its potential as an anxiolytic-like and/or antifatigue agent. In this study, we used socially isolated mice as a high-stress animal model and investigated whether long-term oral administration of L-ornithine had antistress effects on group-housed or socially isolated mice. For this purpose, we used behavioral and pharmacological methods including the elevated plus maze test, the open field test, the stress-induced hyperthermia test, and the pentobarbital-induced sleep test. In group-housed mice, no effect of L-ornithine treatment was observed in any behavioral test. In the isolated mice, however, the treatment decreased locomotor activity and increased the time spent in the central circle in the open field test. In addition, it increased sleep duration in the pentobarbital-induced sleep test. Performance in the elevated plus maze test and stress-induced hyperthermia test was not affected by the treatment. These findings suggest that long-term oral administration of L-ornithine ameliorates antistress effects on highly stressed socially isolated mice.