As an important coarse inorganic fraction of soil, gravel may regulate the effects of the interaction between above- and belowground communities and affect the relationship between microorganisms and plants in alpine ecosystems. However, comparatively little is known about the effects of gravel on the acquisition pattern of nitrogen (N) by microorganisms and plants in alpine ecosystems. In this study, a 15N-labelling experiment was conducted to investigate the acquisition pattern of organic (15N-glycine) and inorganic N (15N-NO3− and 15N-NH4+) by microorganisms and plants under three particle sizes of gravel mulch (fine: 2–10 mm, medium: 10–20 mm, coarse: 20–40 mm) on a semiarid Tibetan grassland. Gravel mulch significantly improved the 15N recovery of Stipa purpurea, but had no significant impacts on A. nanschanica. Therefore, gravel mulch decreased the ratio of microbial biomass 15N recovery to plant biomass 15N recovery for S. purpurea, but caused little effect on the state of N competition between plants and soil microbes for A. nanschanica. The N absorption preference of plants from both species shifted from an individual preference for 15N-NO3− in the natural (i.e., control) microplots to a common preference for 15N-NO3−and 15N-NH4+ in the fine- and medium-sized gravel mulch microplots, while there were no significant differences in microbial N recovery between 15N-NO3− and 15N-NH4+ across all treatments. The results helped to improve the understanding of the acquisition pattern of N by microorganisms and plants under the influence of gravel mulch in alpine ecosystems, and provide theoretical support for revegetation in alpine ecosystems in the future.