Terrestrial ecosystems worldwide are receiving increasing amounts of biologically reactive nitrogen (N) as a consequence of anthropogenic activities. This intended or unintended fertilization can have a wide-range of impacts on biotic communities and hence on soil respiration. Reduction in below-ground carbon (C) allocation induced by high N availability has been assumed to be a major mechanism determining the effects of N enrichment on soil respiration. In addition to increasing available N, however, N enrichment causes soil acidification, which may also affect root and microbial activities. The relative importance of increased N availability vs. soil acidification on soil respiration in natural ecosystems experiencing N enrichment is unclear. We conducted a 12-year N enrichment experiment and a 4-year complementary acid addition experiment in a semi-arid Inner Mongolian grassland. We found that N enrichment had contrasting effects on root and microbial respiration. N enrichment significantly increased root biomass, root N content and specific root respiration, thereby promoting root respiration. In contrast, N enrichment significantly suppressed microbial respiration likely by reducing total microbial biomass and changing the microbial community composition. The effect on root activities was due to both soil acidity and increased available N, while the effect on microbes primarily stemmed from soil acidity, which was further confirmed by results from the acid addition experiment. Our results indicate that soil acidification exerts a greater control than soil N availability on soil respiration in grasslands experiencing long-term N enrichment. These findings suggest that N-induced soil acidification should be included in predicting terrestrial ecosystem C balance under future N deposition scenarios.