Both acute brain injuries and neurodegenerative diseases are accompanied by neuroinflammation. The outcome of neuroinflammation and the prognosis of brain functional status depend on the balance of pro-inflammatory and anti-inflammatory factors. Many studies are aimed at finding possible therapeutic targets allowing to shift inflammatory response processes towards anti-inflammatory mechanisms. It has been shown that channels formed by pannexin proteins are expressed in all brain cells including astrocytes. However, their role in the processes of neuroinflammation is still unclear. Channels formed by pannexin 1 (Panx1) may be involved in proinflammatory activation of astrocytes induced by thrombin and/or lipopolysaccharide (LPS). The aim of this study was to assess thrombin- and LPS-induced activation of primary mouse cortical astrocytes under Panx1 blockade by probenecid. Functional profile of astrocytes, their proliferation and secretory activity changed both in case of thrombin application (50 nM and 100 nM) and in case of incubating cells with LPS. The observed increasing of nitric oxide (NO), β-hexosaminidase HEX and IL6 secretion stopped after the cells were treated with probenecid. Based on the obtained results, probenecid can be considered as a potential agent influencing the inflammatory process in brain tissue by stabilizing astrocytes through inactivation of Panx1 and reduction of astrogliosis.