The eukaryote Tree of Life (eToL) depicts the relationships among all eukaryotic organisms; its root represents the Last Eukaryotic Common Ancestor (LECA) from which all extant complex lifeforms are descended1. Locating this root is crucial for reconstructing the features of LECA, both as the endpoint of eukaryogenesis and the start point for the evolution of the myriad complex traits underpinning the diversification of living eukaryotes. However, the position of the root remains contentious due to pervasive phylogenetic artefacts stemming from inadequate evolutionary models, poor taxon sampling and limited phylogenetic signal1. Here we estimate the root of the eToL with unprecedented resolution on the basis of a new, much larger, dataset of mitochondrial proteins that includes all known eukaryotic supergroups. Our analyses of a 100 taxon × 93 protein dataset with state-of-the-art phylogenetic models and an extensive evaluation of alternative hypotheses show that the eukaryotic root lies between two multi-supergroup assemblages: ‘Opimoda+’ and ‘Diphoda+’. This position is consistently supported across different models and robustness analyses. Notably, groups containing ‘typical excavates’ are placed on both sides of the root, suggesting the complex features of the ‘excavate’ cell architecture trace back to LECA. This study sheds light on the ancestral cells from which extant eukaryotes arose and provides a crucial framework for investigating the origin and evolution of canonical eukaryotic features. The root of the eukaryote Tree of Life is estimated from a new, larger dataset of mitochondrial proteins including all known eukaryotic supergroups, showing it lies between two multi-supergroup assemblages.