The complex interplay of biochemical signaling and mechanical traction forces regulate the position of cellular nuclei. Although the phenomenon of nuclear rotation has been observed for many years, the influence of substrate elasticity was unknown. We discovered another layer of complexity to this phenomenon: nuclear rotation is dependent on substrate elasticity. Nuclear rotation is drastically reduced on physiologically relevant stiffnesses. Here, we studied nuclear rotation in mouse C2C12 myoblasts cultured on soft substrates designed to mimic resting tissue (∼26 kPa) and on hard glass substrates. We examined the roles of the actin and microtubule cytoskeleton on the presence and dynamics of nuclear rotation in these two different microenvironments. We demonstrated the clear dependence of nuclear rotation dynamics on matrix stiffness. These results will have important implications for the design of future studies of nuclear rotation and our understanding of the phenomenon as a whole. Unnaturally, hard substrates do not only fail to mimic the in vivo microenvironment, but can also induce cellular processes that would not normally occur in the natural cellular environment.