INTRODUCTION: Management of intractable childhood constipation is still challenging. The efficacy of retrograde colonic enema (RCE) with fecal microbiota transplantation (FMT) in intractable childhood constipation has not been established, although both have demonstrated potential in gastrointestinal diseases. The aim of this study was to determine the safety and efficacy of RCE-based FMT in the treatment of intractable constipation in children. METHODS: A randomized, double-blind, controlled trial with 110 children was conducted. The patients were randomly assigned to the FMT with RCE group or the placebo with RCE group. All participants received a daily RCE, followed by a 4-week FMT treatment (twice a week) and a 12-week follow-up period. Spontaneous bowel movements ≥ 3 per week were the main outcomes, and the risk ratio with 95% confidence interval (CI) was calculated. Changes in intestinal bacterial profile were analyzed by BOX-PCR-based DNA fingerprinting and sequencing. The adverse effects were assessed based on symptoms. RESULTS: At the end of the follow-up period, 22 patients (40.0%) in the FMT with RCE group and 10 patients (18.2%) in the placebo with RCE group had ≥ 3 spontaneous bowel movements per week (net difference = 21.8%, 95% CI: 13.2%-30.4%; risk ratio: 1.364, 95% CI: 1.063-1.749; P < 0.05). Both RCE and FMT enriched the intestinal bacterial diversity of patients with constipation. The adverse events were all mild self-limiting gastrointestinal symptoms. DISCUSSION: FMT enhances the efficacy of RCE, and the use of RCE-based FMT is a safe and effective method in the treatment of intractable constipation in children.