Rapid urban expansion has profound impacts on global biodiversity through habitat conversion, degradation, fragmentation, and species extinction. However, how future urban expansion will affect global biodiversity needs to be better understood. We contribute to filling this knowledge gap by combining spatially explicit projections of urban expansion under shared socioeconomic pathways (SSPs) with datasets on habitat and terrestrial biodiversity (amphibians, mammals, and birds). Overall, future urban expansion will lead to 11–33 million hectares of natural habitat loss by 2100 under the SSP scenarios and will disproportionately cause large natural habitat fragmentation. The urban expansion within the current key biodiversity priority areas is projected to be higher (e.g., 37–44% higher in the WWF’s Global 200) than the global average. Moreover, the urban land conversion will reduce local within-site species richness by 34% and species abundance by 52% per 1 km grid cell, and 7–9 species may be lost per 10 km cell. Our study suggests an urgent need to develop a sustainable urban development pathway to balance urban expansion and biodiversity conservation.