Double-stranded RNA (dsRNA)-dependent protein kinase (PKR), is a serine/threonine kinase that exerts its own phosphorylation and the phosphorylation of the a subunit of the protein synthesis initiation factor eIF-2a. Lysine residues Lys-60, Lys-150, and Lys-440 were previously identified as SUMOylation sites in PKR and the triple PKR-SUMO deficient mutant was shown to have reduced PKR activity. There are five SUMO family members, the most studied being SUMO1 and two highly homologous paralogs, SUMO2 and SUMO3. We report that SUMO1 and SUMO3 expression exert differential effects on endogenous PKR localisation and activation. SUMO1 did not alter PKR localisation and resulted alone in PKR and eIF-2a activation. At the opposite, SUMO3 induced the transfer of PKR from the cytoplasm to the nucleus and annihilated PKR and eIF-2a activation upon viral infection or dsRNA transfection. In addition, encephalomyocarditis virus (EMCV) enhanced PKR conjugation to SUMO1 and SUMO3 but only SUMO3 expression accelerated caspase-dependent EMCV-induced PKR degradation that occurs in the nucleus. Furthermore, the higher EMCV-induced PKR activation by SUMO1 was correlated with an inhibition of EMCV. Importantly, PKR activation by SUMO1 in the absence of viral infection suggests a novel mechanism for PKR activation and the shift of PKR to the nucleus by SUMO3, may shed a new light on the possible nuclear PKR functions independently of its activation.