AbstractUrban microbiome plays crucial roles in human health and are related to various diseases. The MetaSUB Consortium has conducted the most comprehensive global survey of urban microbiomes to date, profiling microbial taxa/functional genes across 60 cities worldwide. However, the influence of environmental/demographic factors on urban microbiome remains to be elucidated. We collected 35 environmental and demographic characteristics to examine their effects on global urban microbiome diversity/composition by PERMANOVA and regression models. PM₁₀ concentration was the primary determinant factor positively associated with microbial α-diversity (observed species: p = 0.004, β = 1.66, R² = 0.46; Fisher’s alpha: p = 0.005, β = 0.68, R² = 0.43), whereas GDP per capita was negatively associated (observed species: p = 0.046, β = −0.70, R² = 0.10; Fisher’s alpha: p = 0.004, β = −0.34, R² = 0.22). The β-diversity of urban microbiome was shaped by seven environmental characteristics, including Köppen climate type, vegetation type, greenness fraction, soil type, PM_2.5 concentration, annual average precipitation and temperature (PERMANOVA, p < 0.001, R² = 0.01–0.06), cumulatively accounted for 20.3% of the microbial community variance. Canonical correspondence analysis (CCA) identified microbial species most strongly associated with environmental characteristic variation. Cities in East Asia with higher precipitation showed an increased abundance of Corynebacterium metruchotii, and cities in America with a higher greenness fraction exhibited a higher abundance of Corynebacterium casei. The prevalence of antimicrobial resistance (AMR) genes were negatively associated with GDP per capita and positively associated with solar radiation (p < 0.005). Total pathogens prevalence was positively associated with urban population and negatively associated with average temperature in June (p < 0.05). Our study presents the first comprehensive analysis of the influence of environmental/demographic characteristics on global urban microbiome. Our findings indicate that managing air quality and urban greenness is essential for regulating urban microbial diversity and composition. Meanwhile, socio-economic considerations, particularly reducing antibiotic usage in regions with lower GDP, are paramount in curbing the spread of antimicrobial resistance in urban environments.