Background: Hirschsprung's disease (HSCR) is characterized by missing of enteric neurons in the terminal areas of the whole gut, which is causally related to poor proliferation of enteric neural crest cells (ENCCs). Our aim is to explore how miR-431-5p interacts with its target gene in regulation of proliferation of ENCCs in HSCR. Methods: Mouse model of HSCR was established by Benzalkonium chloride (BAC) treatment. Quantitative Real-time PCR and western blotting were performed to determine the miR-431-5p and the LRSAM1 expression in colon tissues of the HSCR group (n = 8) and the control group (n = 8) and in ENCCs isolated from colon tissues. CCK-8 assay was performed to detect the proliferation of ENCCs of HSCR. ENCCs after transfection with miR-431-5p mimics or miR-431-5p inhibitor. Luciferase reporter assay was conducted to clarify the connections between miR-431-5p and LRSAM1. Results: Upregulation of miR-431-5p and downregulation of LRSAM1 were found in ENCCs of HSCR. Downregulation of miR-431-5p could promote cell proliferation of ENCCs. LRSAM1 was proved to be the target gene of miR-431-5p by luciferase reporter assay. Moreover, proliferation of ENCCs was increased in the miR-431-5p inhibitor group and was suppressed after knocking down LRSAM1. Conclusion: Downregulation of miR-431-5p promoted proliferation of ENCCs via targeting LRSAM1, which provides an innovative and candidate target for treatment of HSCR. © 2019 Japanese Society of Developmental Biologists