Here, we determined the localization and activation of protein kinase B (Akt) in acute cutaneous wound tissue in mice. Akt1 represented the major Akt isoform that was expressed and activated in wound margin keratinocytes and also in the cultured human keratinocyte line HaCaT. Mutation of Akt1 protein, exchanging the activation-essential Ser473 and Thr308 residues for inactive Ala or phosphorylation-mimicking Asp and Glu residues, revealed that phosphorylation of Ser473 represented an essential prerequisite for auto-phosphorylation of Thr308 within the Akt1 protein in keratinocytes. Moreover, cell culture experiments and transfection studies using Thr308 mutated Akt1 proteins demonstrated that phosphorylation of Akt1 at Thr308 appeared to selectively exclude the active kinase from the nucleus and direct the kinase to the cytoplasmic compartment in keratinocytes upon insulin stimulation. In summary, our data show that phosphorylation of Thr308 during insulin-mediated Akt1 activation is an essential prerequisite to exclude Akt1 from the nuclear compartment.