Progressive degeneration of dopaminergic neurons in the substantia nigra (SN) is the underlying cause of Parkinson’s disease (PD). The disease in early stages is difficult to diagnose, because behavioral deficits are masked by compensatory processes. Astrocytic and microglial pathology precedes motor symptoms. Besides supportive functions of astrocytes in the brain, their role in PD is unrecognized. Prolonged dysfunction of astrocytes could increase the vulnerability of dopaminergic neurons and advance their degeneration during aging. The aim of our studies was to find out whether prolonged dysfunction of astrocytes in the SN is deleterious for neuronal functioning and if it influences their survival after toxic insult or changes the compensatory potential of the remaining neurons. In Wistar rat model, we induced activation, prolonged dysfunction, and death of astrocytes by chronic infusion of fluorocitrate (FC) into the SN, without causing dopaminergic neuron degeneration. Strongly enhanced dopamine turnover in the SN after 7 days of FC infusion was induced probably by microglia activated in response to astrocyte stress. The FC effect was reversible, and astrocyte pool was replenished 3 weeks after the end of infusion. Importantly, the prolonged astrocyte dysfunction and microglia activation accelerated degeneration of dopaminergic neurons induced by 6-hydroxydopamine and blocked the behavioral compensation normally observed after moderate neurodegeneration. Impaired astrocyte functioning, activation of microglia, diminishing compensatory capability of the dopaminergic system, and increasing neuronal vulnerability to external insults could be the underlying causes of PD. This animal model of prolonged astrocyte dysfunction can be useful for in vivo studies of glia–microglia–neuron interaction.