Fluoxetine (Flx)-induced neuronal plasticity plays an important role in the effective treatment of depression and mood disorders. It is less understood whether repeated Flx treatment induces astrocytic plasticity that outlasts the presence of the drug in the body. We showed previously that Flx-induced neuronal plasticity in the medial prefrontal cortex (mPFC) persisted up to 20 days after the treatment. In this study, adult rats were subjected to a 15-day repeated Flx treatment at a daily dose of 20 mg/kg body weight. Astrocytic metabolites and markers were assessed in the mPFC at day 1 (d1) and day 20 (d20) after the treatment. Significant transient reductions in the concentrations of astrocytic metabolites taurine and myo-inositol and the expressions of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4) were observed in the mPFC of Flx-treated rats at d1, which recovered to the control levels at d20. Further, Flx treatment resulted in long-lasting changes in Kir4.1 expression in the mPFC, which remained downregulated at d20. The expression of 5-HT receptor in the mPFC of Flx-treated rats was downregulated at d1 but became upregulated at d20. In summary, repeated Flx treatment induces both transient and long-term astrocytic plasticity in the mPFC of adult rats. The changes observed at d1 are consistent with disturbed water homeostasis and astrocytic de-maturation in the mPFC. The persistent changes in the expressions of Kir4.1 and 5-HT at d20, presumably of the astrocytic origin, might have contributed to the long-term neurotrophic effects of repeated Flx treatment in the mPFC.