AbstractSerine/threonine protein phosphatase 1 (PP1) regulates multiple cellular processes. Protein phosphorylation–dephosphorylation is largely altered during ischemia and subsequent reperfusion. The brain is particularly vulnerable to stress resulting from ischemia‐reperfusion (IR), however, the acquisition of ischemic tolerance (IT) protects against IR stress. We studied PP1 complexes in response to IR stress and IT in brain using proteomic characterization of PP1 complexes in animal models of IR and IT. PP1α and PP1γ were immunoprecipitated and resolved by 2‐D. DIGE analysis detected 14 different PP1‐interacting proteins that exhibited significant changes in their association with PP1α or PP1γ. These proteins were identified by MALDI‐TOF MS. Seven had the PP1‐binding RVxF motif. IR altered the interaction of heat shock cognate 71 kDa‐protein, creatine kinase B, and dopamine‐ and cAMP‐regulated phosphoprotein 32 kDa (DARPP32) with both PP1α and PP1γ, and the interaction of phosphodiesterase‐6B, transitional ER ATPase, lamin‐A, glucose‐regulated 78 kDa‐protein, dihydropyrimidinase‐related protein‐2, γ‐enolase, neurofilament‐L, and ubiquitin ligase SIAH2 with PP1γ. IT prevented most of the IR‐induced effects. This study identifies novel PP1α‐ and PP1γ‐interacting proteins and reveals an in vivo modularity of PP1 holoenzymes in response to physiological ischemic stress. It supports a potential role of PP1 in IR stress and as a target of the endogenous protective mechanisms induced by IT.