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Abstract The continuously increasing concentration of atmospheric CO 2 has considerably altered ecosystem functioning. However, few studies have examined the long-term (i.e. over a decade) effect of elevated CO 2 on soil microbial communities. Using 16S rRNA gene amplicons and a GeoChip microarray, we investigated soil microbial communities from a Californian annual grassland after 14 years of experimentally elevated CO 2 (275 ppm higher than ambient). Both taxonomic and functional gene compositions of the soil microbial community were modified by elevated CO 2. There was decrease in relative abundance for taxa with higher ribosomal RNA operon (rrn) copy number under elevated CO 2 , which is a functional trait that responds positively to resource availability in culture. In contrast, taxa with lower rrn copy number were increased by elevated CO 2. As a consequence, the abundance-weighted average rrn copy number of significantly changed OTUs declined from 2.27 at ambient CO 2 to 2.01 at elevated CO 2. The nitrogen (N) fixation gene nifH and the ammonium-oxidizing gene amoA significantly decreased under elevated CO 2 by 12.6% and 6.1%, respectively. Concomitantly, nitrifying enzyme activity decreased by 48.3% under elevated CO 2 , albeit this change was not significant. There was also a substantial but insignificant decrease in available soil N, with both nitrate (NO 3 −) (−27.4%) and ammonium (NH 4 +) (−15.4%) declining. Further, a large number of microbial genes related to carbon (C) degradation were also affected by elevated CO 2 , whereas those related to C fixation remained largely unchanged. The overall changes in microbial communities and soil N pools induced by long-term elevated CO 2 suggest constrained microbial N decomposition, thereby slowing the potential maximum growth rate of the microbial community. Graphical abstract Unlabelled Image Highlights • Effects of 14 years of experimentally elevated CO 2 on soil microbes in a semi-arid grassland were examined. • The abundance-weighted average rrn copy number of significantly changed OTUs declined by elevated CO 2. • The nitrogen fixation gene nifH and the ammonium-oxidizing gene amoA significantly decreased by elevated CO 2. • Elevated CO 2 constrained microbial N decomposition, thereby slowing potential maximum growth rate of microbial community. [ABSTRACT FROM AUTHOR]