Urbanization is among the main drivers of biodiversity loss, altering ecological communities and ecosystem functioning. Understanding how urbanization affects biodiversity is essential for biodiversity conservation in increasingly human-dominated landscapes. Arthropods play key ecological roles and are widely used to assess the impacts of environmental change. Among them, spiders are suitable indicators of urbanization because they are abundant, widespread, and sensitive to habitat alteration. Despite their ecological importance, spider communities remain relatively understudied, largely because the ecology of many species is still poorly known. Here, we present a pioneer study investigating how spider community composition and community functional traits vary along an urbanization gradient. From May to September 2025, spiders were sampled monthly at 19 urban, suburban, and rural sites across the Metropolitan City of Milan. At each site, a 15-m transect was surveyed using standardized active sampling methods and pitfall traps. We analysed spider abundance, species richness, community composition, and functional traits in relation to urbanization and environmental variables. Our results suggest that urbanization negatively affects spider biodiversity. In particular, species richness and diversity declined with increasing levels of urban development, while community composition shifted towards a dominance of a few urban-tolerant taxa, indicating processes of biotic homogenization. In contrast, sites characterized by greater environmental heterogeneity supported higher spider diversity across the urbanization gradient. Community functional traits also varied along the gradient and were associated with environmental heterogeneity, further indicating that urbanization acts as an environmental filter. Together, these findings suggest that maintaining environmental heterogeneity may help mitigate the ecological impacts of urbanization and contribute to biodiversity conservation in urban landscapes.