The Mre11-Rad50-Nbs1 (MRN) complex plays a key role in the DNA damage response, presenting challenges for DNA viruses and retroviruses. To inactivate this complex, adenovirus (Ad) makes use of the E1B-55K and E4-open reading frame 6 (ORF6) proteins for ubiquitin (Ub)-mediated, proteasome-dependent degradation of MRN and the E4-ORF3 protein for relocalization and sequestration of MRN within infected-cell nuclei. Here, we report that Mre11 is modified by the Ub-related modifier SUMO-2 and Nbs1 is modified by both SUMO-1 and SUMO-2. We found that Mre11 and Nbs1 are sumoylated during Ad5 infection and that the E4-ORF3 protein is necessary and sufficient to induce SUMO conjugation. Relocalization of Mre11 and Nbs1 into E4-ORF3 nuclear tracks is required for this modification to occur. E4-ORF3-mediated SUMO-1 conjugation to Nbs1 and SUMO-2 conjugation to Mre11 and Nbs1 are transient during wild-type Ad type 5 (Ad5) infection. In contrast, SUMO-1 conjugation to Nbs1 is stable in cells infected with E1B-55K or E4-ORF6 mutant viruses, suggesting that Ad regulates paralog-specific desumoylation of Nbs1. Inhibition of viral DNA replication blocks deconjugation of SUMO-2 from Mre11 and Nbs1, indicating that a late-phase process is involved in Mre11 and Nbs1 desumoylation. Our results provide direct evidence of Mre11 and Nbs1 sumoylation induced by the Ad5 E4-ORF3 protein and an important example showing that modification of a single substrate by both SUMO-1 and SUMO-2 is regulated through distinct mechanisms. Our findings suggest how E4-ORF3-mediated relocalization of the MRN complex influences the cellular DNA damage response.