The current paradigm of plant nutrient limitation during ecosystem development predicts a change from nitrogen (N) limitation when substrates are young to phosphorus (P) limitation when substrates are old. However, there are surprisingly few direct tests of this model. We evaluated this theory experimentally along a three million year semi-arid substrate age gradient using resource additions to intercanopy spaces dominated by the C4 bunchgrass Bouteloua gracilis. Unlike other gradients in subtropical and temperate ecosystems, soil water availability also increases strongly across this semi-arid system due to finer texture with substrate age. We found that aboveground net primary production (ANPP) of B. gracilis was limited by both water and N on the 55 ky substrate; not limited by N, P, or water on the 750 ky substrate; and limited by P alone on the 3000 ky substrate. Notably, measures of foliar nutrient concentration and N:P mass ratios were unable to predict nutrient limitations in these semi-arid systems. In unamended plots, mean ANPP declined dramatically at 3000 ky compared to the younger substrate age sites, presumably due to progressive limitation by P. This decline in ANPP late in ecosystem development is consistent with a reduction in soil total carbon and N storage at this site and provides a mechanism for successional retrogression in ecosystem structure and function. Our results unify biogeochemical theory across disparate ecosystems while illustrating the important water-nutrient interactions in these semi-arid ecosystems to further define the nature of nutrient limitations in terrestrial ecosystems.