The extracellular matrix (ECM) stiffness has direct effect on cancer cells homeostasis (e.g., cell volume), which is critical for regulation of their migration. However, the relationship among ECM stiffness, cell volume and cancer cell migration in three-dimensional (3D) microenvironment remains elusive. In this work, we prepared the collagen-alginate hydrogels with tunable stiffness to study how the 3D ECM stiffness influences cell volume and their migration. We found the cell volume homeostasis and migration speed of the MDA-MB-231 cells are both regulated by 3D ECM stiffness, while cell migration speed shows the same stiffness-dependent trend with cell volume. Deviating the cell volume from its homeostasis state can cause a significant decrease in its migration ability, which can be recovered through recovering the cell volume to its homeostasis state. This work reveals for the first time that 3D ECM stiffness regulates cell migration behavior through regulating cell volume homeostasis, which may provide a novel view in the exploration of the underlying mechanisms of cancer metastasis and cellular mechanotransduction. (C) 2020 Elsevier Inc. All rights reserved.