Serving as a source of glutathione and up-taking and metabolizing glutamate are the primary supportive role of astrocytes for the adjacent neurons. Despite the clear physical association between astrocytes and α-synuclein, the effect of extracellular α-synuclein on these astrocytic functions has not yet been elucidated. Hence, we aim to assess the effect of various forms of α-synuclein on antioxidant mechanism and glutamate metabolism. Wild-type and A53T/A30P double-mutant α-synuclein, both in monomeric and aggregated forms, were added extracellularly to media of midbrain rat astrocyte culture, with their survival, oxidative, and nitrative stress, glutathione and glutamate content, expression of enzymes associated with oxidative stress and glutamate metabolism, glutamate and glutathione transporters being assessed along with the association/engulfment of these peptides by astrocytes. A30P/A53T peptide associated more with astrocytes, and low-extracellular K+ concentration showed prominent reduction in the engulfment of the monomeric forms, suggesting that the association of the aggregated forms was greater with the membrane. The peptide-associated astrocytes showed lower survival and increased oxidative stress generation, owing to the decrease in nuclear localization of Nrf2 and increase in iNOS, and further aggravated by the decrease in glutathione content and related enzymes like glutathione synthetase, glutathione peroxidase, and glutathione reductase. Glutamate uptake increased in aggregate-treated cells due to the increase in GLAST1 expression, de novo synthesis of glutamate by pyruvate carboxylase, and/or glutamine synthase, bolstered by the differential glutamate dehydrogenase enzyme activity. We thus show for the first time that extracellular α-synuclein exposure leads to astrocytic dysfunction with respect to the antioxidant mechanism and glutamate metabolic profile. The impact was higher in the case of the aggregated and mutated peptide, with the highest dysfunction for the mutant aggregated α-synuclein treatment.