Nitrogen (N) fertilizer and water availability can independently stimulate or limit soil N dynamics through direct and indirect processes. Importantly, soil N mineralization (Nmin) is a major N source for maize but affected by N fertilization and water availability. We examined in-situ net Nmin, soil enzyme activity, and maize N uptake in a semiarid region of North America in response to two levels of water availability (100% and 70% crop evapotranspiration, ET) and three levels of N fertilization (22–275 kg ha− 1 capturing low, optimal, and excess N fertilization. Nitrogen mineralization rates peaked relatively early in the growing season leading to asynchrony between soil N supply and plant demand. Later in the season when plant N uptake was highest, Nmin rates were high under low N with full water supply, and high under high N with limited water supply, resulting in an N fertilizer and water interaction. Soil L-leucine amino peptidase (LAP) and β-1,4-N-acetyl-glucosaminidase (NAG), which can be indicators of gross Nmin, increased with N fertilizer additions but were not affected by water supply. Further research is needed to understand the mechanisms underlying this interaction as well as exploring if gross Nmin has a similar response. Maize N uptake increased with N fertilizer additions under both levels of water availability but was higher in the full water supply. In the limited water availability, increased plant N uptake with increased N fertilization did not translate to large grain yield increases highlighting the impact of water stress, especially during grain fill.