The effect of nanotopographies on cell adhesion, migration, proliferation, differentiation, and/or apoptosis have been studied over the last two decades. However, the effect of nanotopography on gene transfection of adhered cells is far from understood. One key phenomenon of using nanotopography is mimicry of native cell morphology in vitro such as in alignment of skeletal myoblasts on nanogrooves. The formation of focal adhesions, the cytoskeleton, and the morphology of cell nuclei are altered by underlying nanogrooves, but the role of these changes in gene transfection are not well understood. In this study, C2C12 skeletal myoblasts were transfected using polyethylenimine (PEI)/DNA complexes on nanogrooved patterns of two groove widths (400 and 800 nm) at three depths (50 nm and 400 or 500 nm). The results showed that the deep nanogrooved surfaces (i.e., 400/400 and 800/500) induced formation of aligned, parallel F-actin and elongated nucleus morphology. Gene transfection was also reduced on the deep nanogrooved surfaces. Disruption of F-actin organization using Cytochalasin D (Cyto-D) restored the nuclear morphology accompanied by higher transfection efficiency, demonstrating that the reduction in gene expression on deep nanogrooves was due to cytoskeletal stretching and nucleus elongation. Spatiotemporal images of fluorescent-labeled PEI/DNA complexes showed that endocytosis of PEI/DNA complexes was retarded and DNA trafficking into the cell nucleus was reduced. This study demonstrates for the first time the important role of cytoskeletal organization and nuclear morphology in PEI-mediated gene transfection to skeletal myoblasts using nanogrooved patterns. These findings are informative for in vitro studies and could potentially be useful in in vivo intramuscular (IM) administration.