BACKGROUND: Serotonin is strongly involved in the regulation of brain development, including the proper formation of neuronal circuits and synaptic plasticity. One of the factors that can affect brain serotonin levels is exposure to fluoxetine (FLX), a selective serotonin reuptake inhibitor, the first-line pharmacological treatment for depression and anxiety in the pediatric population. The safety of early-life FLX treatment is still questionable. Women are more prone to anxiety and depression from a young age. We hypothesized that juvenile FLX treatment influences the brain maturation and behavior of adolescent females. METHODS: On postnatal days 20 to 28, juvenile female rats were injected once daily with FLX. Five days later, anxiety- and fear-related behaviors and amphetamine-induced locomotor activity were assessed. On postnatal day 40, the numbers of neurons and glial cells in the medial prefrontal cortex (mPFC) and hippocampus were estimated via stereological methods. Additionally, the mRNA expression of cell survival/apoptosis and synaptic plasticity markers was evaluated via RT‒qPCR. RESULTS: FLX-treated females showed decreased anxiety level, freezing behavior during fear conditioning and amphetamine-induced locomotor activity when compared to control females. Simultaneously, FLX-injected females presented greater regional volume and numbers of neurons and astrocytes in specific subregions of the mPFC when compared to the control group. Additionally, FLX-treated females showed increased expression of genes regulating cell survival and reduced mRNA levels of AMPA glutamate receptors in the mPFC. CONCLUSIONS: Juvenile FLX affects the maturation of the mPFC and attenuates anxiety-like behavior, fear memory and the locomotor response to amphetamine in adolescent females.