This article provides the first checklist of the spider fauna of the Marakele National Park, including their conservation status and level of endemicity based on their known distribution. Standard collecting methods were used to sample spiders during a survey in February 2010. Thirty-six families that include 100 genera and 135 species were recorded. Salticidae (19 spp.), Araneidae (18 spp.) and Thomisidae (17 spp.) were the most species-rich families, while 14 families were only represented by a single species. About 6 % of the South African spider fauna and 14% of the Limpopo Province fauna are protected in the park; 21 species are South African endemics, but only one (Cheiracanthium foordi Lotz, 2015) is a Marakele National Park endemic.