Abstract Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, but use of these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix would differentiate into enteric neural subsets capable of inducing neurotransmitter-mediated smooth muscle cell (SMC) contraction and relaxation. NPCs were isolated from the appendix and small intestine (SI) of rabbits. Bioengineered internal anal sphincter constructs were developed using the same source of smooth muscle and innervated with NPCs derived from either the appendix or SI. Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry, and functionality was assessed through force-generation studies. Expression of neural and glial differentiation markers was observed in constructs containing appendix- and SI-derived NPCs. The addition of acetylcholine to both appendix and SI constructs caused a robust contraction that was decreased by pretreatment with the neural inhibitor tetrodotoxin (TTX). Electrical field stimulation caused relaxation of constructs that was completely abolished in the presence of TTX and significantly reduced on pretreatment with nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride [l-NAME]). These data indicate that in the presence of identical soluble factors arising from intestinal SMCs, enteric NPCs derived from the appendix and SI differentiate in a similar manner and are capable of responding to physiological stimuli. This coculture paradigm could be used to explore the nature of the soluble factors derived from SMCs and NPCs in generating specific functional innervations. Significance This study demonstrates the ability of neural stem cells isolated from the appendix to differentiate into mature functional enteric neurons. The differentiation of neural stem cells from the appendix is similar to differentiation of neural stem cells derived from the gastrointestinal tract. The appendix is a vestigial organ that can be removed with minimal clinical consequence through laparoscopy. Results presented in this paper indicate that the appendix is a potential source of autologous neural stem cells required for cell therapy for the gastrointestinal tract.