cisTEM is user-friendly software to process cryo-EM images of macromolecular complexes and obtain high-resolution 3D reconstructions from them. It was originally developed by Tim Grant, Alexis Rohou and Nikolaus Grigorieff and comprises a number of tools to process image data including movies, micrographs and stacks of single-particle images, implementing a complete “pipeline” of processing steps to obtain high-resolution single-particle reconstructions. cisTEM is distributed under the Janelia Research Campus Software License and can be downloaded here. We recommend downloading and using the pre-compiled binaries, rather than compiling the source code, for best performance. New users are encouraged to follow the tutorial, which provides a quick way to become familiar with the most important functions of cisTEM.