Abstract Agricultural large-scale land acquisitions have been linked with enhanced deforestation and land use change. Yet the extent to which transnational agricultural large-scale land acquisitions (TALSLAs) contribute to—or merely correlate with—deforestation, and the expected biodiversity impacts of the intended land use changes across ecosystems, remains unclear. We examine 178 georeferenced TALSLA locations in 40 countries to address this gap. While forest cover within TALSLAs decreased by 17% between 2000 and 2018 and became more fragmented, the spatio-temporal patterns of deforestation varied substantially across regions. While deforestation rates within initially forested TALSLAs were 1.5 (Asia) to 2 times (Africa) higher than immediately surrounding areas, we detected no such difference in Europe and Latin America. Our findings suggest that, whereas TALSLAs may have accelerated forest loss in Asia, a different mechanism might emerge in Africa where TALSLAs target areas already experiencing elevated deforestation. Regarding biodiversity (here focused on vertebrate species), we find that nearly all (91%) studied deals will likely experience substantial losses in relative species richness (−14.1% on average within each deal)—with mixed outcomes for relative abundance—due to the intended land use transitions. We also find that 39% of TALSLAs fall at least partially within biodiversity hotspots, placing these areas at heightened risk of biodiversity loss. Taken together, these findings suggest distinct regional differences in the nature of the association between TALSLAs and forest loss and provide new evidence of TALSLAs as an emerging threat to biodiversity in the Global South.