Enteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies following successful colonization of recipient gut after transplantation. However, the ability of ENSC to rescue pathophysiological conditions remains unclear. Interestingly, loss of neuronal subtypes, including neuronal nitric oxide synthase (nNOS), has been implicated in enteric neuropathies. nNOS-/- mice display slow colonic transit providing a model to test ENSC rescue in a pathological setting. Our aim was to assess the functional integration of transplanted ENSC within recipient nNOS-/- colon. Donor ENSC were obtained from Wnt1-cre;YFP transgenic mice for fluorescent labeling and fate mapping of cells. Integration and functionality were assessed using immunolabeling and in vivo and organ bath physiology utilizing electrical field stimulation (EFS). After 1 month, transplanted cell networks were identified in recipient nNOS-/- colon with spread of 5.19±0.5 mm2;n=8. YFP+/nNOS+ neurons were identified and transcriptional analysis showed specific expression of nNOS in recipient nNOS-/- colon. In vivo analysis showed significant recovery in total gastrointestinal (GI) transit time in transplanted nNOS-/- (114.8±3.6 mins,n=5), similar to C57BL/6J controls (117.4±2.6 mins,n=5), compared to non-transplanted nNOS-/- (177±8.15mins,n=5;P=0.0001). In NANC (non-adrenergic non-cholinergic) conditions, organ bath physiology revealed significant increases in EFS-induced relaxation (Area under curve;AUC) in transplanted nNOS-/- (-1.13±0.16g.s,n=5) compared with non-transplanted nNOS-/- (-0.31±0.0.08g.s,n=5;P=0.0016). In transplanted colonic segments, addition of the nitric oxide synthase blocker L-NAME resulted in significant reductions in the observed EFS-induced relaxation (-0.74±0.17g.s vs -0.12±0.16g.s,n=4;P=0.0389) demonstrating restoration of nitrergic responses after ENSC transplantation. In addition to partial restoration of nitrergic responses significant increases in basal contractile amplitude were observed in transplanted nNOS-/- colonic segments (0.30±0.06g,n=5) compared with both C57BL/6J (0.10±0.01g,n=5;P=0.0093) and non-transplanted nNOS-/- mice (0.05±0.008g,n=5;P=0.0025). Interestingly these high amplitude contractions were unaffected by application of tetrodotoxin, suggesting that transplantation of ENSC can also lead to potential changes in underlying myogenic motility patterns. Our experiments show, for the first time, that transplanted ENSC not only integrate but effect restoration of function, at the organ level, in a pathological GI disease model. This recovery of function appears to be associated with both ENSC-specific and non ENSC-specific processes. Ongoing work is targeting the non-specific processes associated with ENSC transplantation including possible modification of the transplanted cellular microenvironment.