Abstract： Objective:To construct a regulatory network of Hirschsprung's disease (HSCR) through bioinformatics.Methods:RNA sequencing was utilized for detecting the expression profiles of long non-coding RNA (lncRNA) and mRNA in HSCR and normal colon tissues. A co-expression network of lncRNA-mRNA was constructed with the mRNA expression data from the database of GSE96854. Three lncRNAs and mRNAs were selected for validation using quantitative polymerase chain reaction (QPCR).Results:A total of 224 lncRNAs were identified by RNA sequencing, including 108 up-regulated and 116 down-regulated lncRNAs; 785 differentially expressed genes (DEGs) were identified with 82 up-regulated and 703 down-regulated. DEGs were mainly enriched in KEGG pathways such as cGMP-PKG and NF-κB signaling pathways. In the GSE96854 dataset, 1216 significantly up-regulated and 1376 significantly down-regulated DEGs were identified. There were 178 common DEGs between two datasets with 175 significantly down-regulated and only 3 significantly up-regulated DEGs. Protein-protein interaction (PPI) analysis revealed that SNAP25, SYP, ATP2B3 and ZAP70 were core interactive genes in the network, especially SNAP25. A total of 146 lncRNA-mRNA regulatory networks were identified. AC074286.1-ADRA2A-cGMP-PKG and DUX4L9-ATP2B3-NF-κB signaling pathways were identified as lncRNA-mRNA pathway regulatory networks. QPCR results revealed a significant down-regulation of ADRA2A, ATP2B3, TNFSF14, AC074286.1, DUX4L9 and REXO1L1P, consistent with the results of RNA sequencing.Conclusion:LncRNA and mRNA molecular markers are closely involved in inflammation, apoptosis and neural-related processes of HSCR.