The intracellular signaling mechanisms that couple transient cerebral ischemia to cell death and neuroprotective mechanisms provide potential therapeutic targets for cardiac arrest. Protein phosphatase (PP)-1 is a major serine/threonine phosphatase that interacts with and dephosphorylates critical regulators of energy metabolism, ionic balance, and apoptosis. We report here that PP-1I, a major regulated form of PP-1, is activated in brain by approximately twofold in vivo following cardiac arrest and resuscitation in a clinically relevant pig model of transient global cerebral ischemia and reperfusion. PP-1I purified to near homogeneity from either control or ischemic pig brain consisted of the PP-1 catalytic subunit, the inhibitor-2 regulatory subunit, as well as the novel constituents 14-3-3γ, Rab GDP dissociation protein β, PFTAIRE kinase, and C-TAK1 kinase. PP-1I purified from ischemic brain contained significantly less 14-3-3γ than PP-1I purified from control brain, and purified 14-3-3γ directly inhibited the catalytic subunit of PP-1 and reconstituted PP-1I. These findings suggest that activation of brain PP-1I following global cerebral ischemia in vivo involves dissociation of 14-3-3γ, a novel inhibitory modulator of PP-1I. This identifies modulation of PP-1I by 14-3-3 in global cerebral ischemia as a potential signaling mechanism-based approach to neuroprotection.