During skeletal muscle development, correct cellular orientation is vital to generate desired longitudinal contraction for functional muscle fibres. In this reported study, submicron-imprint lithography was used to generate submicron-grooved surfaces on polystyrene plates to induce striated myotubes in vitro. Mouse muscle myoblast cells cultured on a submicron-grooved surface migrated faster in a directionally uniform fashion; in comparison, cells cultured on a flat surface grew and migrated slower in indiscriminate directions. Subsequent maturation of the myoblast cells formed along the submicron-groove surface resulted in a tandem of parallel myotubes that were both longer and greater in circumference than in the case of the flat surface. In a functional test, the co-culture submicron-groove-grown myotubes with neurotransmitter secreting cells further demonstrated contraction abilities, suggesting submicron-groove-guided growth served to enhance myotube formation while retaining striated motifs and physiological functionality for muscle tissue engineering.