Background: In recent years, emerging researches have shown that circular RNA play critical regulatory roles in a variety of diseases. However, the biogenesis, regulation, function and mechanism of circRNAs in Hirschsprung's disease remain largely unknown. Methods: qRT-PCR was used to determine the expression of circANKRD12/circTIMMDC1 in HSCR tissues. Transwell and wound healing assay were conducted to assess the role of circANKRD12/circTIMMDC1 in neural cell migration. Dual-luciferase reporter assay, RIP assay and RNA pull-down assay were performed to evaluate the direct interaction between circANKRD12/circTIMMDC1 and miR-181b-5p and alleviate suppression on target PROX1 expression. Results: The expression of circANKRD12/circTIMMDC1 and PROX1 were down-regulated in HSCR tissues compared with control tissues, while the miR-181b-5p, NOTCH1, HES1 expression were up-regulated. Knockdown of circANKRD12 and circTIMMDC1 synergically inhibited the migration of human neural cells, whereas overexpression of circANKRD12 and circTIMMDC1 had the opposite effects. Transfection of miR-181b-5p inhibitor or mimics reversed the effects of circANKRD12/circTIMMDC1 on cell migration. We demonstrated that circANKRD12/circTIMMDC1 acted as “molecular sponge” of miR-181b-5p to rescue the repressive effect of miR-181b-5p on its target PROX1, and suppressed neural cell migration through activating NOTCH1-HES1 signaling pathway. Conclusions: Our research reveals a novel negative regulatory loop circANKRD12/circTIMMDC1-miR-181b-5p-PROX1-NOTCH1-HES1, in the pathogenesis of HSCR providing an exploitable biomarker and therapeutic targets for HSCR.