Brain edema formation following brain injury is a serious but still poorly treatable medical condition. The understanding of volume regulation in astrocytes, the main cells involved in the formation of cytotoxic brain edema, is key for the development of novel treatment strategies. This study investigates the role of potassium-chloride cotransporters (KCC) for cell volume regulation in glial cells. PCR revealed the expression of KCC isoforms in a glial cell line (C6) and primary cultured astrocytes. Specific inhibition of KCCs caused glial cell swelling and resulted in a complete inhibition of regulatory volume decrease upon hypotonic medium-induced cell swelling. Therefore, our results show that KCCs play an important role in the maintenance and regulation of cell volume in astrocytes.