The MNI-HISUB25 dataset contains manual hippocampal subfield labels and associated high-resolution MRI data (submillimetric T1- and T2-weighted images, detailed sequence information, and stereotaxic probabilistic anatomical maps) based on 25 healthy subjects. Data were acquired on a 3-Tesla MRI system using a 32 phased-array head coil. The protocol divided the hippocampal formation into three subregions: subicular complex, Cornu Ammonis 1, 2 and 3 (CA1-3), and CA4-dentate gyrus (CA4-DG). Segmentation was guided by consistent intensity and morphology characteristics of the densely myelinated molecular layer together with few geometry-based boundaries flexible to overall mesiotemporal anatomy, and achieved excellent intra-/inter-rater reliability (Dice index ≥90/87%). The dataset can inform neuroimaging assessments of the mesiotemporal lobe and help to develop segmentation algorithms relevant for basic and clinical neurosciences.