The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Initial efforts to purify and characterise XPF-ERCC1 used negative stain electron microscopy and involved the addition of an EM-visible label to mark the C-terminal tail of XPF. Subsequently, cryo-electron microscopy structures of both label-free DNA-free and DNAbound human XPF-ERCC1 were solved. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks ERCC1 DNA-binding elements and restricts access to the XPF catalytic site. Binding of a model DNA junction separates the XPF helical and ERCC1 (HhH)2 domains, promoting activation. Using these structural data, we propose a model for a 5’-NER incision complex involving XPF-ERCC1-XPA and a DNA junction substrate. Our mutational data suggest xeroderma pigmentosum patient mutations compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations display substantial in vitro activity but are resistant to activation by ICLR recruitment factor SLX4. In addition, a low-resolution structure of the functionally uncharacterised (XPFERCC1) 2 heterotetramer was solved indicating substantial intrinsic flexibility. These data provide insights into XPF-ERCC1 architecture, catalytic activation and mechanisms underlying patient mutations.