Objective To identify the conditions for co-culturing embryonic rat spinal motoneurons and C2C12 myotubes, establish a stable co-culture system, and to form functional neuromuscular junction in vitro. Methods The C2C12 myoblasts were cultured to 60%-70% confluence and then were induced by differentiation medium. The embryonic spinal cord anterior horn motor neurons were obtained from 15-16 d pregnant SD rats, and were implanted in the myotubes after differentiating for 5 days; the products were co-cultured in the basic serum-free culture medium Neurobasal+2% B27. The neuronal morphology and projection length at each stage, myotube morphological and contraction characteristics, and formation of the neuromuscular junction were observed under an inverted microscope. The α-bungarotoxin (α-BTX), which can specifically bind to acetylcholine receptor (AChR) of the postsynaptic membrane, was examined by immunofluorescence technique and the muscle contraction in the co-culture system was recorded by screen recording technology. Results Both the primal spinal motoneurons and the C2C12 myotubes survived in the co-culture system, with further differentiation and maturation. On day 3 the axons extended to the myotube membrane surface or surrounded the myotubes. On day 7 the myotubes were arranged in the same direction, with wide rhythmic contraction, and immunofluorescence showed that α-BTX specifically bound to AChR of the postsynaptic membrane. On day 10 of co-culture, the motor neurons began to have apoptosis and the myotube cells gradually shrank. Conclusion Under in vitro culture condition, motor neurons and skeletal muscle cells can co-exist and grow, establishing synaptic connections, triggering a series of neuromuscular junction signal transduction, and causing rhythmic contraction of the myotubes.