Previously we found that prolonged endoplasmic reticulum (ER) stress caused by coxsackievirus B3 (CVB3) infection led to p58IPK downregulation and subsequent cell apoptosis. This finding implies that p58IPK expression benefits cell survival and counteracts CVB3-induced apoptosis. In testing this hypothesis, we first found that PI3K/Akt survival pathway is more sensitive than ERK1/2 in response to p58IPK expression. This finding was further verified by silencing p58IPK with specific siRNAs, which led to the significant suppression of phosphorylation of Akt (p-Akt) but not ERK1/2. Further, using CVB3-infected cell line expressing dominant negative ATF6a (DN-ATF6a), we found that expression of p58IPK and p-Akt was significantly reduced, which led to the decreased cell viability. However, when the DN-ATF6a cells were transiently transfected with p58IPK, an opposite result was obtained. Finally, by CVB3 infection of cells stably expressing p58IPK, we found that CVB3-induced mitochondria-mediated apoptosis was suppressed, which was evidenced by the reduced cytochrome c release and upregulation of the mitochondrial membrane protein mitofusin 2. However, silencing p58IPK with either specific siRNAs or DN-ATF6a sensitized cells to CVB3-induced apoptosis. These results suggest that p58IPK suppresses CVB3-induced apoptosis through selective activation of PI3K/Akt pathway that requires activation of ATF6a and subsequently upregulates mitofusin 2.