Objective To explore the rule of distribution and the expression significance of neuronal nitric oxide synthase (nNOS), paired box 3(Pax3) and connexin 43(Cx43) proteins in the early developing spinal cord of human embryos. Methods During the 5th to 16th weeks of human gestation, the expression productions of nNOS, Pax3 and Cx43 proteins were investigated in the anterior horn of spinal cord using the immunohistochemical methods. Results During the 5th to 16th weeks of human gestation, the immunohistochemical staining intensities of both nNOS and Pax3 in the anterior horn neurons of spinal cord increased from weakly to strongly positive staining. During the 5th to 12th weeks of human gestation, the immunohistochemical negative staining of Cx43 protein was observed in the anterior horn neurons of spinal cord, while the positive staining was observed from the 13th to 16th weeks.Conclusion nNOS, Pax3 and Cx43 proteins are related to the growth and development of spinal cord in human embryos.