Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a serine/threonine kinase that exerts its own phosphorylation and the phosphorylation of the α subunit of the protein synthesis initiation factor eIF-2α. PKR was identified as a target of SUMOylation and the triple PKR-SUMO deficient mutant on Lysine residues K60-K150-K440 has reduced PKR activity. We report that SUMO1 and SUMO3 expression exert differential effects on PKR localization, activation and stability. SUMO1 or SUMO3 did not alter the repartition of PKR in the cytoplasm and the nucleus. However, in SUMO3-expressing cells PKR was found more concentrated around the perinuclear membrane and was recruited from small speckles to nuclear dots. Interestingly, SUMO1 expression alone resulted in PKR and eIF-2α activation, whereas SUMO3 reduced PKR and eIF-2α activation upon viral infection or dsRNA transfection. In addition, encephalomyocarditis virus (EMCV) enhanced PKR conjugation to SUMO1 and SUMO3 but only SUMO3 expression promoted caspase-dependent EMCV-induced PKR degradation. Furthermore, the higher EMCV-induced PKR activation by SUMO1 was correlated with an inhibition of EMCV. Importantly SUMO1, by inducing PKR activation in the absence of viral infection, and SUMO3, by counteracting both PKR activation and stability upon viral infection, shed a new light on the differential effects of SUMO-modified PKR.