Hirschsprung's disease (HSCR) is a polygenic disease, of which the cause remains to be elucidated. It has been suggested that SRY-related HMG-box 9 (SOX9) is fundamental for the correct development of oligodendrocytes and astrocytes; however, not the development of neurons. There are currently no reports regarding SOX9 expression in patients with HSCR; therefore, the present study aimed to investigate the expression of microRNA-124 (miR-124) and its target gene, SOX9, in HSCR. Quantitative polymerase chain reaction (qPCR), western blot analysis and immunohistochemistry were used to detect the mRNA and protein expression of miR-124 and SOX9 in patients with HSCR. miR-124 expression was observed to be markedly higher in stenotic colon segment tissues compared with normal colon segment tissues in patients with HSCR. Furthermore, mRNA and protein analyses revealed that SOX9 expression was also higher in the stenotic colon segment tissues compared with the normal colon segment tissues. In conclusion, these data suggest that miR-124 and its target gene, SOX9, are overexpressed in the stenotic colon segment of patients with HSCR, and may have a significant role in the development of HSCR.