<p>Wastewater treatment plants (WWTPs) are essential infrastructure for protecting water resources in rapidly urbanizing regions. However, current knowledge of the impacts of urbanization on microbial communities in WWTPs remains ambiguous. Here we show that urbanization and operational conditions jointly shape microbial diversity and homogeneity in WWTPs. By analysing 934 samples from 217 WWTPs across the globe, we found that urbanization factors such as city population size and economic development are strongly associated with changes in community similarity. Population size is likely associated with increased similarity among communities, while socio-economic development shows the opposite association. In contrast, operational conditions, including sludge retention time and substrate concentration, present a relatively high explanation degree for microbial community richness. In particular, we observed that homogenized communities and a loss of diversity correlate closely with performance and stability. This study provides a global-scale assessment of how urbanization and operational factors interact to shape microbial communities in WWTPs.</p>