The unique high-altitude environmental factors in the Linzhi region of Tibet have not been fully investigated in terms of their impact on the microbial mechanisms of water ecology, for which we explored the response of activated sludge microorganisms to different hydraulic retention times (HRT) based on 16S rRNA gene sequencing and PICRUST2 functional prediction. The results showed that different HRT had significant effects on Phylum, Class, Genus, Species and OTU levels, as well as on diversity, richness and evenness (P < 0.05). The relative abundance of Bacteroidetes, Chloroflexi, Firmicutes and AAP99 in the dominant bacterial phylum and genus was significantly influenced by different HRT. In addition, we identified the denitrification reaction as the major pathway in nitrogen metabolism in this study, as well as most of the significantly enriched genera were denitrifying genera as determined by LEfSe analysis. The overall low relative abundance of the nitrifying bacteria genera AOB and NOB may also be an important reason for the poor nitrogen removal. The effect of HRT on the expression of genes of the five functional modules of nitrogen metabolism was inconsistent. In conclusion, the above studies further complement the studies on the effects of plateau-specific environments on the microbial mechanisms of water ecology under different HRT conditions.