The entorhinal cortex (EC) plays a pivotal role in memory functions and spatial navigation, connecting the hippocampus with the neocortex. This brain region integrates a wide range of cortical and subcortical inputs and it is one of the first brain regions affected by Alzheimer´s disease. However, its synaptic organization in the human brain is largely unknown due to the difficulties involved in studying the human brain via electron microscope techniques. Here, we used Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) to perform a 3D analysis of the synapses in all layers of the medial EC from human brain autopsies with very short post-mortem delays. Using this technology, 12,974 synapses were 3D reconstructed at the ultrastructural level. We studied several synaptic parameters, including the synaptic density, 3D spatial distribution, the proportion of synaptic types (excitatory and inhibitory), as well as the size of each synaptic junction, the synaptic shape and postsynaptic elements.The EC presented a distinct set of synaptic features, differentiating this region from other human cortical areas. Furthermore, synaptic organization within the EC was predominantly homogeneous, although layers I and VI exhibited several synaptic characteristics that were distinct from other layers. The present study constitutes an extensive description of the synaptic organization of the neuropil of all layers of the EC, a crucial step to better understand the connectivity of this cortical region, in both health and disease.3D reconstruction of a dendritic segment from FIB/SEM serial images, in which excitatory (green) and inhibitory (red) synapses are established.