We introduce DiffFit, a differentiable algorithm for fitting protein atomistic structures into experimental Cryo-Electron Microscopy (cryo-EM) volume maps. This process is crucial in structural biology for reconstructing large meso-scale models of complex protein assemblies and complete cellular structures. Unlike current methods requiring manual fitting followed by automated fine-tuning, DiffFit enables automatic fitting with visual inspection and interactive revision. Our approach employs differentiable 3D rigid transformations and a novel loss function based on multi-resolution volume arrays and negative space exploitation. Evaluations on three use cases using realistic datasets demonstrate that DiffFit significantly outperforms previous methods. DiffFit is available as an open-source plugin (github .