Nitrogen (N) cycling was analyzed in the Kalahari region of southern Africa, where a strong precipitation gradient (from 978 to 230 mm mean annual precipitation) is the main variable affecting vegetation. The region is underlain by a homogeneous soil substrate, the Kalahari sands, and provides the opportunity to analyze climate effects on nutrient cycling. Soil and plant N pools, 15N natural abundance (δ15N), and soil NO emissions were measured to indicate patterns of N cycling along a precipitation gradient. The importance of biogenic N2 fixation associated with vascular plants was estimated with foliar δ15N and the basal area of leguminous plants. Soil and plant N was more 15N enriched in arid than in humid areas, and the relation was steeper in samples collected during wet than during dry years. This indicates a strong effect of annual precipitation variability on N cycling. Soil organic carbon and C/N decreased with aridity, and soil N was influenced by plant functional types. Biogenic N2 fixation associated with vascular plants was more important in humid areas. Nitrogen fixation associated with trees and shrubs was almost absent in arid areas, even though Mimosoideae species dominate. Soil NO emissions increased with temperature and moisture and were therefore estimated to be lower in drier areas. The isotopic pattern observed in the Kalahari (15N enrichment with aridity) agrees with the lower soil organic matter, soil C/N, and N2 fixation found in arid areas. However, the estimated NO emissions would cause an opposite pattern in δ15N, suggesting that other processes, such as internal recycling and ammonia volatilization, may also affect isotopic signatures. This study indicates that spatial, and mainly temporal, variability of precipitation play a key role on N cycling and isotopic signatures in the soil–plant system.