Picornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER-resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2alpha, but infection with UV-inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2alpha was dependent on PKR: eIF2alpha phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR-eIF2alpha pathway. Pulse-chase experiments revealed that EV71 infection inhibited translation and activation of ATF6. Expression of BiP at the protein level was activated by a virus-dependent, ATF6-independent mechanism. EV71 upregulated XBP1 mRNA level, but neither IRE1-mediated XBP1 splicing nor its active spliced protein was detected, and its downstream gene, EDEM, was not activated. Epigenetic BiP overexpression alleviated EV71-induced ER stress and reduced viral protein expression and replication. Our results suggest that EV71 infection induces ER stress but modifies the outcome to assist viral replication.