The absorption of iron is influenced by numerous dietary and physiological factors. We have previously demonstrated that zinc treatment of intestinal cells increases iron absorption via induction of the apical membrane iron transporter divalent metal ion transporter-1 (DMT1). To better understand the mechanisms of zinc-induced iron absorption, we have studied the effect of zinc on iron uptake, iron transporter and iron regulatory protein (IRP 1 and 2) expression and the impact of the PI3K pathway in differentiated Caco-2 cells, an intestinal cell culture model. We found that zinc induces DMT1 protein and mRNA expression. Zinc-induced DMT1 expression and iron absorption were inhibited by siRNA silencing of DMT1. Furthermore, zinc treatment led to increased abundance of IRP2 protein in cell lysates and in polysomal fractions, implying its binding to target mRNAs. Zinc treatment induced Akt phosphorylation, indicating the activation of the PI3K pathway. LY294002, a specific inhibitor of PI3K inhibited zinc-induced Akt phosphorylation, iron uptake, DMT1 and IRP2 expression. Furthermore, LY294002 also decreased the basal level of DMT1 mRNA but not protein expression. siRNA silencing of IRP2 led to down-regulation of both basal and zinc-induced DMT1 protein expression, implying possible involvement of post-transcriptional regulatory mechanisms. In agreement with these findings, zinc treatment stabilized DMT1 mRNA levels in actinomycin D-treated cells. Based on these findings, we conclude that zinc-induced iron absorption involves elevation of DMT1 expression by stabilization of its mRNA, by a PI3K/IRP2-dependent mechanism.