Hirschsprung's disease (HSCR) is a complex developmental defect characterized by the absence of enteric ganglia in the gastrointestinal tract. Although the genetic defect of enteric nervous system (ENS) was identified to play a critical role in the progress of HSCR, the systemic genetic dissection of HSCR still needs to be clarified. In this study, we firstly performed exome sequencing of two HSCR patients from a Han Chinese family, including the affected mother and son. After the initial quality filtering (coverage ≥ 5X and SNP quality score ≥ 40) of the raw data, we identified 13,948 and 13,856 single nucleotide variants (SNVs), respectively. We subsequently compared the SNVs against public databases (dbSNP130, HapMap, and 1000 Genome Project) and obtained a total of 15 novel nonsynonymous SNVs in 15 genes, which were shared between these two patients. Follow-up Sanger sequencing and bioinformatics analysis highlighted variant c.853G>A (p.E285K) in NRG3, a gene involved in the development of ENS. In the validation phase, we sequenced all nine exons of NRG3 in 96 additional sporadic HSCR cases and 110 healthy individuals and identified another nonsynonymous variant c.1329G>A (p.M443I) and two synonymous variants c.828G>A (p.T276T) and c.1365T>A (p.P455P) only in the cases. Our results indicated that NRG3 may be a susceptibility gene for HSCR in a Chinese population.