Objective: To investigate the effect of intestinal flora in children with functional constipation (FC) on expression of acid-sensitive Ion channel 3(ASIC3) in rats and their regulation in intestinal motility. Methods: Faeces of FC children identified according to RomeⅣ criteria and healthy children from the First Affiliated Hospital of Anhui Medical University from December 2017 to June 2018 were collected, and then made into fecal microbiota solution.A pseudo - sterile rat model was established, according to the random number table method, and the rats were randomly divided into the treatment group and the control group, with 12 rats in each group, then the treatment group was given fecal microbiota solution of the children with FC and the control group was given fecal microbiota solution of the healthy children.The visceral sensitivity and intestinal propulsion rate of rats were determined by means of abdominal withdrawal reflex (AWR), while the intestinal microorganism of rats and children with FC were determined by 16SrDNA high-throughput sequencing, and the expressions of ASIC3 of intestinal in mRNA and protein were determined by adopting fluorescence quantitative PCR and Western blot. Results: The species and quantity of intestinal flora of the children with FC and rats implanted with FC faecal bacteria were reduced(all P<0.05), and firmicutes and bacteroidetes were the main bacteria; compared to the control group, the small intestine propulsion rate(52% vs.74%) and visceral sensitivity(78 mmHg vs.63 mmHg) of the treated group were significantly decreased compared with those in the control group (all P<0.05); the mRNA (0.003 1±0.000 8 vs.0.012 4±0.002 5) and protein levels of ASIC3 (0.013 2±0.001 9 vs.0.072 1±0.008 7) in the small intestine were down-regulated significantly(all P<0.05); and the mRNA (0.002 8±0.000 7 vs.0.009 4±0.001 1) and protein levels of ASIC3(0.038 2±0.004 5 vs.0.089 7±0.009 4) in the colon were down-regulated significantly(all P<0.05). Conclusions Children with FC have intestinal flora disorder, and intestinal flora of FC children may affect intestinal motility by down-regulating the expression of intestinal ASIC3 in rats.