Urbanization represents the extreme case of human influence on an ecosystem. Biogeochemical cycling of nitrogen (N) in cities is very different from that of non-urban landscapes due to the large input of reactive forms of N and the heterogeneous distribution of various land uses that alters landscape connections. To quantify the likely effects of human activities on soil N and other soil properties in urban ecosystems, we conducted a probability-based study to sample 203 plots randomly distributed over the 6,400 km2 Central Arizona-Phoenix Long-Term Ecological Research (CAP LTER) area, which encompasses metropolitan Phoenix with its 3.5 million inhabitants. Soil inorganic N concentrations were significantly higher in urban residential, non-residential, agricultural, transportation, and mixed sites than in the desert sites. Soil water content and organic matter were both significantly higher under urban and agricultural land uses, whereas bulk density was lower compared to undeveloped desert. We calculated that farming and urbanization on average had caused an accumulation of 7.23 g m−2 in soil inorganic N across the CAP study area. Average soil inorganic N of the sampled desert sites (3.23 g m−2) was much higher than the natural background level reported in the literature. Laboratory incubation studies showed that many urban soils exhibited net immobilization of inorganic N, whereas desert and agricultural soils showed small, but positive, net N mineralization. The large accumulation of inorganic N in soils (mostly as nitrate) was highly unusual in terrestrial ecosystems, suggesting that in this arid urban ecosystem, N is likely no longer the primary limiting resource affecting plants, but instead poses a threat to surface and groundwater contamination, and influences other N cycling processes such as denitrification.