Acetylcholinesterase (AChE) inhibitors have protective and anti-inflammatory actions against brain injury, mediated by nicotinic α7 cholinergic receptor activation. The use of AChE inhibitors in patients is limited by systemic cholinergic side effects. Posiphen, a stereoisomer of the AChE inhibitor Phenserine, lacks AChE inhibitor activity. The purpose of this study is to determine the protective effect of Posiphen in cellular and animal models of stroke. Both Posiphen and Phenserine reduced glutamate-mediated neuronal loss in co-cultures of primary cortical cells and microglia. Phenserine-, but not Posiphen-, mediated neuroprotection was diminished by the nicotinic α7 receptor antagonist methyllycaconitine. Posiphen antagonized NMDA-mediated Ca influx, thapsigargin-mediated neuronal loss and ER stress in cultured cells. Early post-treatment with Posiphen reduced ER stress signals, IBA1 immunoreactivity, TUNEL and infarction in the ischemic cortex, as well as neurological deficits in stroke rats. These findings indicate that Posiphen is neuroprotective against stroke through regulating Cai and ER stress.