We describe a three-dimensional magnetic twisting technology that is useful in characterizing the mechanical properties of cells. Using three pairs of orthogonally-aligned coils, an oscillatory mechanical torque was applied to magnetic beads about any chosen axis, at frequencies up to 1 kHz. The cell deformation was measured in response to a torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half of that parallel to the long axis. Elongated cells on poly-L-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and of the nucleolus were sensitive to loading directions, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating structural pathways of mechanotransduction.