Iron is the fundamental element for numerous physiological functions. Plasmalemmal divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of ferrous (Fe), whereas transferrin receptors (TFR) carry transferrin (TF)-bound ferric (Fe). In this study we performed detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca]) in astrocytes. Administration of Fe or Fe in μM concentrations evoked [Ca] in astrocytes in vitro and in vivo. Iron ions trigger increase in [Ca] through two distinct molecular cascades. Uptake of Fe by DMT1 inhibits astroglial Na-K-ATPase, which leads to elevation in cytoplasmic Na concentration, thus reversing Na/Ca exchanger and thereby generating Ca influx. Uptake of Fe by TF-TFR stimulates phospholipase C to produce inositol 1,4,5-trisphosphate (InsP), thus triggering InsP receptor-mediated Ca release from endoplasmic reticulum. In summary, these findings reveal the mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload.