The dramatic increased rates of uncontrolled urbanization in various parts of the World have resulted in loss of native species and overall threats to biodiversity. Over the last few decades Saudi Arabia has witnessed a remarkably rapid population growth and unparalleled levels of urbanization, leading to threats to biodiversity. Ants were pitfall-trapped across an urban-rural gradient to evaluate ant assemblage responses to urbanization in Wadi Hanifa, Riyadh, Central Saudi Arabia. Fifteen sampling sites were selected along three different urbanization gradients, each traversing urban, suburban and rural zones. Within each site 10 traps were distributed and operated for 7 consecutive days, at 3-monthly intervals throughout one year. Vegetation, ground cover, and chemical and physical soil variables at sampling sites were analyzed concurrently. Ant abundance, species richness, evenness, and diversity indices of Shannon and Simpson were calculated for each site using PC-ORD to demonstrate diversity patterns along the urbanization gradients. Ant assemblages were assessed by detrended corresponding analysis (DCA), canonical correspondence analysis (CCA), and analysis of similarity (ANOSIM) using PC-ORD. Indicator species analysis was conducted to define representative species along the urbanization gradient. A total of 42 ant species were identified. The diversity parameters were consistent across the urbanization gradient. However, significant differences were observed in the ant assemblages between rural and urban, suburban and urban, but only marginal between rural and suburban. Eleven ant species were identified as indicator species (IV values between 50.7-80.7%). The ant assemblages were influenced by flora, ground cover, and soil variables.