An annotated checklist and taxonomic review of the spiders of Saint Helena is presented. A total of 88 genera and 114 species are known; of these, 12 genera and 45 species are endemic. Two new genera and two new species are described: Anapistula martinae Sherwood, Harvey, Fowler, Joshua, Stevens, Scipio-O'Dean & Ellick sp. nov., Helenidion Sherwood, Marusik, Fowler, Stevens & Joshua gen. nov., Ischnothyreus christyjoae Sherwood, Henrard, Peters, Stevens & Fowler sp. nov., and Trust Sherwood, Marusik, Wilkins, P. Ashmole & M. Ashmole gen. nov. Three new combinations are proposed: Helenidion sciaphilum (Benoit, 1977) comb. nov. (type species), Helenidion huberti (Benoit, 1977) comb. nov., and Zelotes funereus (Dalmas, 1921) comb. nov. Lepthyphantes albimaculatus (O. Pickard-Cambridge, 1873) (Linyphiidae) is synonymized with Lepthyphantes leprosus (Ohlert, 1865) syn. nov. Lynxosa Roewer, 1960 gen. rest. is restored to house the lycosids L. inexorabilis (O. Pickard-Cambridge, 1870) comb. rest., L. ligata (O. Pickard-Cambridge, 1870) comb. rest., L. nefasta (Tongiorgi, 1977) comb. nov., and L. veseyensis (Sherwood, Henrard, Logunov & Fowler, 2023) comb. nov. The previously undescribed sexes of Benoitodes sanctaehelenae (Strand, 1909) (Gnaphosidae), Hahnia laticeps Simon, 1898 (Hahniidae), Helenactyna crucifera (O. Pickard-Cambridge, 1873) (Dictynidae, first known male for the genus), Paraheliophanus napoleon Clark & Benoit, 1977 (Salticidae), and Trust solium (Benoit, 1977) comb. nov. (Theridiidae) are described. Bonapruncinia sanctaehelenae Benoit, 1977, originally described from juveniles, is supplemented with a first description of the adult female. Lectotypes and paralectotypes are designated for the salticid species Paraheliophanus subinstructus (O. Pickard-Cambridge, 1873) and Pellenes inexcultus (O. Pickard-Cambridge, 1873). Seven species are newly recorded from the island: Clubiona hitchinsi Saaristo, 2002 (Clubionidae), Latrodectus renivulvatus Dahl, 1902 (Theridiidae), Micropholcus fauroti (Simon, 1887) (Pholcidae), Oecobius marathaus Tikader, 1962 (Oecobiidae), Steatoda nobilis (Thorell, 1875) (Theridiidae), Theridion melanostictum O. Pickard-Cambridge, 1876 (Theridiidae), and Theridion proximum Lawrence, 1964 (Theridiidae). Epigynal duplication, a rare anomaly in spiders, is reported for the prodidomid Zimirina relegata Cooke, 1977.