Living cells respond to their mechanical microenvironments during development, healing, tissue remodeling and homeostasis attainment. However, this mechanosensitivity has not yet been established definitively for cells in three-dimensional (3D) culture environments, in part because of challenges associated with providing uniform and consistent 3D environments that can deliver a large range of physiological and pathophysiological strains to cells. Here, we report microscale magnetically actuated, cell-laden hydrogels (μMACs) for investigating the strain-induced cell response in 3D cultures. μMACs provide high-throughput arrays of defined 3D cellular microenvironments that undergo reversible, relatively homogeneous deformation following non-contact actuation under external magnetic fields. We present a technique that not only enables the application of these high strains (60%) to cells but also enables simplified microscopy of these specimens under tension. We apply the technique to reveal cellular strain-threshold and saturation behaviors that are substantially different from their 2D analogs, including spreading, proliferation, and differentiation. μMACs offer insights for mechanotransduction and may also provide a view of how cells respond to the extracellular matrix in a 3D manner. Micromechanical strain is shown to play a key role in the growth and development of tissue and muscle cells in three-dimensional environments. Feng Xu and co-workers have developed an innovative medium for visualizing and influencing cell growth with biocompatible, water-based hydrogels. Using photolithography, the team fabricated a micrometre-thick, multilayer device in which cell-laden hydrogels sat above a separate hydrogel film infused with magnetic iron nanoparticles. Applying a magnetic field caused the lower hydrogel film to deform homogenously, altering the strain of the three-dimensional cell-growth region by up to 60 per cent. This method has the advantage that it does not use damaging mechanical contacts. The transparent hydrogels enable real-time microscopy imaging of fibroblast and myoblast viability and proliferation under realistic strain conditions — valuable data for artificial muscle applications and pathophysiological studies of injury healing. We report microscale magnetically actuated, cell-laden hydrogels (μMACs) for investigating the strain-induced cell response in three-dimensional (3D) microenvironments. These μMACs provide high-throughput arrays of defined 3D cellular microenvironments that undergo reversible, relatively homogeneous deformation following non-contact actuation under external magnetic fields. Such technique not only enables the application of high strains (up to 60%) to cells but also enables simplified microscopic visualization of these specimens under tension. The μMACs offer insights for mechanotransduction and may also provide a view of how cells respond to extracellular matrix in 3D.