Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an alpha6beta4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3zeta, we demonstrate that in keratinocytes 14-3-3zeta/tau heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3zeta acts as a dominant negative and inhibits the interaction between 14-3-3tau and SSH1. These results implicate 14-3-3zeta/tau heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.