S100B is an astrocytic cytokine that has been shown to be involved in several neurodegenerative diseases. We used an astrocytoma cell line (U373 MG) silenced for S100B, and stimulated it with amyloid beta-peptide (Aβ) as a known paradigm factor for astrocyte activation, and showed that the ability of the cell (including the gene machinery) to express S100B is a prerequisite for inducing reactive astrocytic features, such as ROS generation, NOS activation and cytotoxicity. Our results showed that control astrocytoma cell line exhibited overexpression of S100B after Aβ treatment, and subsequently cytotoxicity, increased ROS generation and NOS activation. In contrast, cells silenced with S100B were essentially protected, consistently reducing cell death, significantly decreasing oxygen radical generation and nitric oxide synthase activity. The conclusive aim of the present study was to show a causative linkage between the cell expression of S100B and induction of astrocyte activation processes, such as cytotoxicity, ROS and NOS activation.