Docosahexaenoic acid (DHA) has neuroprotective effects in several neurodegenerative disease conditions. However, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on astrocyte Ca(2+) signaling under in vitro ischemic conditions (oxygen/glucose deprivation and reoxygenation, OGD/REOX). OGD (2h) triggered a Ca(2+) (ER) store overload (∼1.9-fold). Ca(2+) uptake by the Ca(2+) (ER) stores was further augmented during REOX and Ca(2+) (ER) was elevated by ∼4.7-fold at 90min REOX. Interestingly, Ca(2+) (ER) stores abruptly released Ca(2+) at ∼120min REOX and emptied at 160min REOX. Depletion of Ca(2+) (ER) stores led to delayed elevation of intracellular Ca(2+) concentration (Ca(2+) (cyt) ) and cell death. Activation of the purinergic receptor P2Y1 was responsible for the release of Ca(2+) (ER) . Most importantly, DHA blocked the initial Ca(2+) (ER) store overload, the delayed depletion of Ca(2+) (ER) , and rise in Ca(2+) (cyt) , which was in part via inhibiting d-myo-inositol 1,4,5-triphosphate receptors. The DHA metabolite DiHDoHE exhibited similar effects. DHA also attenuated expression of phosphorylated eukaryotic initiation factor 2α and activating transcription factor-4, two ER stress markers, following in vitro ischemia. Taken together, these findings suggest that DHA has protective effects in astrocytes following in vitro ischemia, in part, by inhibiting Ca(2+) dysregulation and ER stress.