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Human land use such as pastoralism may be a key determinant of wildlife populations. We studied the influence of land use on wildlife and livestock in south-western Kalahari, Botswana, during the wet and dry seasons. We included two types of livestock areas and two types of wildlife protection areas. Wild and domestic mammals > 0.2 kg were sampled by distance technique. Small mammals 0.2 kg were trapped and a vegetation survey was performed. Livestock and some medium-sized wildlife species were most abundant in livestock areas, while large-sized wildlife species were mainly restricted to wildlife areas. Most red-listed species were confined to the national park. The influence of land use on abundances changed moderately between seasons. However, cattle moved into wildlife areas during the wet season. Differences in species richness and diversity were most pronounced during the dry season. The trapping frequency of small mammals was highest in the national park. Differences in vegetation structure included sparser field layer and higher cover of an invasive shrub in livestock areas. Our study shows the importance of protected areas for many wildlife species in the Kalahari. It also shows the value of a multi-species approach for investigating the effects of land use on wildlife communities.