Double-stranded DNA coming from, for example, viruses, bacteria, or apoptotic cells is recognized by the cGAS-STING signaling pathway comprising the cyclic GMP-AMP synthase (cGAS) and the stimulator of interferon genes (STING) receptors. The pathway induces type I interferon response and activates transcription of interferon-stimulated genes and proinflammatory cytokines. Though the brain is an immune-privileged site, the blood-brain barrier (BBB) elicits inflammatory immune response in neurodegenerative diseases. Parkinson's disease is characterized by α-synuclein oligomer (αSO) aggregates, neurodegeneration, and mitophagy, which potential can activate the cGAS-STING pathway. Here, we studied the cGAS-STING pathway in a co-culture model of the rat BBB treated with and without α-synuclein monomers (αSM) or oligomers (αSO). Activation of the cGAS-STING pathway did not change barrier integrity and junctional protein staining, but it induced the transcription of the interferon-stimulated gene Viperin and the proinflammatory cytokine tumor necrosis factor-α in brain endothelial cells. Furthermore, STING activation increased the protein level of Viperin in astrocytes. The treatment with αSO, but not αSM, decreased barrier tightness and induced the transcription of Viperin and tumor necrosis factor-α in brain endothelial cells. In astrocytes, αSO treatment increased not only Viperin and tumor necrosis factor-α mRNA levels, but also interleukin-1β and interleukin-6. In conclusion, cGAS-STING pathway and downstream immune signaling pathways can be activated in the cells of a co-culture model of the BBB without influencing barrier integrity. However, αSO disrupts the BBB integrity and activates the cGAS-STING immune pathway in brain endothelial cells and astrocytes supporting the idea of using cGAS-STING as a therapeutic target in neuroinflammation.