Adolescence is one of the most important periods of brain development in mammals. There is increasing evidence that some medicines during this period can affect brain and behavioral functions in adulthood. Silymarin (SM), a mixture of flavonolignans extracted from the milk thistle Silybum marianum, is known as a hepatoprotective, anti-inflammatory, and neuroprotective drug. Although researchers have extensively studied the effects of SM during adulthood, to date there is no information on the effects of this drug during the stages of brain development on behavioral functions in adulthood. In the current study, we investigated the effects of adolescent SM treatment on body weight and anxiety-like behaviors in adult male and female mice. Adolescent NMRI mice (postnatal day 30–50) were treated orally with water or SM (50 and 100 mg/kg). Animals were weighed during drug treatment and were then subjected to open field, elevated plus maze, and light–dark box tests from postnatal day 70. The results indicated that adolescent SM treatment increased anxiety-like behaviors in open field, elevated plus maze, and light–dark box in adult mice, while not altering body weight. Collectively, these findings suggest that adolescent SM treatment may have profound effects on the development of brain and behavior in adulthood.