This paper describes a fabrication method of muscle tissue constructs driven by neurotransmitters released from activated motor neurons. The constructs consist of three-dimensional (3D) free-standing skeletal muscle fibers co-cultured with motor neurons. We differentiated mouse neural stem cells (mNSCs) cultured on the skeletal muscle fibers into neurons that extend their processes into the muscle fibers. We found that acetylcholine receptors (AChRs) were formed at the connection between the muscle fibers and the neurons. The neuron–muscle constructs consist of highly aligned, long and matured muscle fibers that facilitate wide contractions of muscle fibers in a single direction. The contractions of the neuron–muscle construct were observed after glutamic acid activation of the neurons. The contraction was stopped by treatment with curare, an neuromuscular junction (NMJ) antagonist. These results indicate that our method succeeded in the formation of NMJs in the neuron–muscle constructs. The neuron–muscle construct system can potentially be used in pharmacokinetic assays related to NMJ disease therapies and in soft-robotic actuators.