The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form. Here, we uncovered that the C-terminal domain of Upf1, conserved in higher eukaryotes and containing several essential phosphorylation sites, also inhibits the flanking helicase domain. With different biochemical approaches we show that this domain, named SQ, directly interacts with the helicase domain to impede ATP hydrolysis and RNA unwinding. The phosphorylation sites in the distal half of the SQ domain are not directly involved in this inhibition. Therefore, in the absence of multiple binding partners, Upf1 is securely maintained in an inactive state by two intramolecular inhibition mechanisms. This study underlines the tight and intricate regulation pathways required to activate multifunctional RNA helicases like Upf1.