Mammalian 14-3-3 isoforms exist predominantly in the brain and are heavily involved in neurological diseases. However, the isoform-specific role of 14-3-3 proteins in the brain remains largely unclear. Here, we investigated the role of 14-3-3 isoforms in rat brains after transient middle cerebral artery occlusion and reperfusion. 14-3-3 beta, eta, gamma and zeta but not epsilon or tau were selectively upregulated in cerebral cortical neurons after ischemia-reperfusion (I/R). Selectively, 14-3-3 beta, gamma and zeta were translocated from cytoplasm into the nuclei of neurons after I/R. 14-3-3 bound to p65 and suppressed p65 expression in N2a cells. In the brain, 14-3-3 could either colocalize with p65 in the nuclei of neurons or segregate from p65 expression in cortical neurons after I/R. All evidence together suggests that 14-3-3 isoforms are differentially induced to enter into the nuclei of neurons after I/R, which might regulate NF kappa B signaling directly or indirectly. Since 14-3-3 proteins are essential for cell survival and NF kappa B is a key transcriptional factor, our data suggest that the 14-3-3/p65 signaling pathway might be a potential therapeutic target for stroke.