The subcellular location of annexin A1 (ANXA1) determines the ultimate fate of neurons after ischemic stroke. ANXA1 nuclear translocation is involved in neuronal apoptosis after cerebral ischemia, and extracellular ANXA1 is also associated with regulation of inflammatory responses. As the factors and mechanism that influence ANXA1 subcellular translocation remain unclear, studies aiming to determine and clarify the role of ANXA1 as a cell fate 'regulator' within cells are critically needed. In this study, we found that intracerebroventricular injection of the recombinant adenovirus vector Ad-S100A11 (carrying S100A11) strongly improved cognitive function and induced robust neuroprotective effects after ischemic stroke in vivo. Furthermore, upregulation of S100A11 protected against neuronal apoptosis induced by oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. Surprisingly, S100A11 overexpression markedly decreased ANXA1 nuclear translocation and subsequently alleviated OGD/R-induced neuronal apoptosis. Notably, S100A11 exerted its neuroprotective effect by directly binding ANXA1. Importantly, S100A11 directly interacted with ANXA1 through the nuclear translocation signal (NTS) of ANXA1, which is essential for ANXA1 to import into the nucleus. Consistent with our previous studies, ANXA1 nuclear translocation after OGD/R promoted p53 transcriptional activity, induced mRNA expression of the pro-apoptotic Bid gene, and activated the caspase-3 apoptotic pathway, which was almost completely reversed by S100A11 overexpression. Thus, S100A11 protects against cell apoptosis by inhibiting OGD/R-induced ANXA1 nuclear translocation. This study provides a novel mechanism whereby S100A11 protects against neuronal cells apoptosis, suggesting the potential for a previously unidentified treatment strategy in minimizing apoptosis after ischemic stroke.