The h5Seurat file format is specifically designed for the storage and analysis of multi-modal single-cell and spatially-resolved expression experiments, for example, from CITE-seq or 10X Visium technologies. It holds all molecular information and associated metadata, including (for example) nearest-neighbor graphs, dimensional reduction information, spatial coordinates and image data, and cluster labels. We also support rapid and on-disk conversion between h5Seurat and AnnData objects, with the goal of enhancing interoperability between Seurat and Scanpy.