Crowding within the cell is optimized to accelerate reactions, but not excessively impede diffusion. However, the mechanisms that regulate crowding are unknown. We developed genetically encoded multimeric (GEM) nanoparticles to study the physical properties of the cytoplasm. GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. We used this system to discover signaling pathways that modulate crowding in yeast and mammalian cells. We found that the mTORC1 pathway tunes macromolecular crowding through regulation of ribosome concentration and thereby regulates the effective diffusion of macromolecules larger than 16 nm in diameter but has no effect on the diffusion of molecules at the 5 nm length-scale, thus providing a mechanism to differentially tune reactions in the cell based on particle size. This tuning of rheology makes induction of a stress response gene more robust to osmotic pressure. Our results connect a central regulator of growth and metabolism to the biophysical properties of the cell.