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Permafrost-underlain tundra soils in Northern Hemisphere are one of the largest reservoirs of terrestrial carbon, which are highly sensitive to microbial decomposition due to climate warming. However, knowledge about the taxonomy and functions of microbiome residing in different horizons of permafrost-underlain tundra soils is still limited. Here we compared the taxonomic and functional composition of microbiome between different horizons of soil cores from a moist tussock tundra ecosystem in Council, Alaska, using 16S rRNA gene and shotgun metagenomic sequencing. The composition, diversity, and functions of microbiome varied significantly between soil horizons, with top soil horizon harboring more diverse communities than sub-soil horizons. The vertical gradient in soil physico-chemical parameters were strongly associated with composition of microbial communities across permafrost soil horizons; however, a large fraction of the variation in microbial communities remained unexplained. The genes associated with carbon mineralization were more abundant in top soil horizon, while genes involved in acetogenesis, fermentation, methane metabolism (methanogenesis and methanotrophy), and N cycling were dominant in sub-soil horizons. The results of phylogenetic null modeling analysis showed that stochastic processes strongly influenced the composition of the microbiome in different soil horizons, except the bacterial community composition in top soil horizon, which was largely governed by homogeneous selection. Our study expands the knowledge on the structure and functional potential of microbiome associated with different horizons of permafrost soil, which could be useful in understanding the effects of environmental change on microbial responses in tundra ecosystems.