The Brazilian Cerrado is the second largest biome in Latin America, extending over more than 200 million ha and hosts some of the most intensive agricultural activities for grain and beef production in the world. Because of the biodiversity richness and high levels of endemism, Cerrado is considered one of world's hotspot for biodiversity conservation. The objectives of this study are three-fold: to present a comprehensive division of Cerrado into different ecoregions that reflect the environmental heterogeneity within the biome; to analyze the ecoregions in terms of biophysical characteristics, protected areas, environmental liability in riparian permanent protection areas along watercourses, and priorities for biodiversity conservation; and to rank the ecoregions in terms of endangerment for biodiversity conservation and restoration. A previous study that delineated 22 ecoregions using geomorphology, vegetation, soil, geology, and plant diversity maps was revised using topography, vegetation, precipitation, and soil maps. Our new ecoregion map consists of 19 units that are unique in terms of landscape characteristics and has been adjusted to the current official boundary map of Cerrado. Some of the ecoregions consist of only one geomorphological compartment, whereas others are heterogeneous, consisting of up to eight compartments. Ferralsols comprise the dominant soil type in 14 of the ecoregions. The percentage of protected areas within ecoregions ranges from 1.7% to 51.5%. The most endangered ecoregion, where land use change critically threatens habitat integrity, is the Depressão Cárstica do São Francisco (states of Bahia, Minas Gerais, and Piauí), where environmental liability along riparian permanent protection areas amounts to 85.6% of the total area. Our proposed ecoregion map provides a spatial framework for regional and local assessments to improve decision-making processes to reconcile conservation and restoration planning, sustainable agriculture, and provision of ecosystem services. Besides de adjustment of the previous Cerrado's ecoregion map to the official biome boundary (relevant for the implementation of public policies of conservation as those regulated by the Brazilian Forest Code), the new analyses of the ecoregion map represent a substantial improvement in comparison to the ones conducted by the previous study in 2003. In addition, current web resources allow us to make all the information used or derived from this study available to other users. This opens the possibility of additional improvements of our findings by the scientific community or to be used effectively by decision makers.