Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2alpha are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2alpha. Functionally, sumoylation reduced the transcriptional activity of HIF-2alpha. Similar to HIF-1alpha, HIF-2alpha is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2alpha during hypoxia but did not affect the total level of HIF-2alpha. The ubiquitin E3 ligases von Hippel-Lindau and RNF4 control the levels of sumoylated HIF-2alpha, indicating that sumoylated HIF-2alpha is degraded via SUMO-targeted ubiquitin ligases.