UNLABELLED: High-throughput microscopy data require a diversity of analytical approaches. However, the construction of workflows that use algorithms from different software packages is difficult owing to a lack of interoperability. To overcome this limitation, we present CellH5, an HDF5 data format for cell-based assays in high-throughput microscopy, which stores high-dimensional image data along with inter-object relations in graphs. CellH5Browser, an interactive gallery image browser, demonstrates the versatility and performance of the file format on live imaging data of dividing human cells. CellH5 provides new opportunities for integrated data analysis by multiple software platforms. AVAILABILITY: Source code is freely available at www.github.com/cellh5 under the GPL license and at www.bioconductor.org/packages/release/bioc/html/rhdf5.html under the Artistic-2.0 license. Demo datasets and the CellH5Browser are available at www.cellh5.org. A Fiji importer for cellh5 will be released soon.