The SUMO pathway parallels the classical ubiquitinylation pathway with three discrete steps: activation involving the enzyme E1, conjugation involving the E2 enzyme UBC9, and substrate modification through the cooperative association of UBC9 and E3 ligases. We report here that the adenoviral protein Gam1 inhibits the SUMO pathway by interfering with the activity of E1 (SAE1/SAE2). In vivo, Gam1 expression leads to SAE1/SAE2 inactivation, both SAE1/SAE2 and UBC9 disappearance, and overall inhibition of protein sumoylation. This results in transcriptional activation of some promoters and is directly linked to inhibition of sumoylation of the transcriptional activators involved. Our results identify a mechanism for interfering with the SUMO pathway and with transcription that could have an impact in the design of novel pharmaceutical agents. They also point out once again to the extraordinary ability of eukaryotic viruses to interfere with the biology of host cells by targeting fundamental biochemical processes.