Type 1 diabetes mellitus (T1DM) is characterized by autoimmune destruction of pancreatic β cells. Previous study has discovered that probiotic strains residing in the gut play essential roles in host immune regulation. However, few clinical results demonstrated probiotic would actually benefit in attenuating glycated hemoglobin (HbA1c) along with inflammatory cytokine levels of the T1DM patients and analyzed their gut microbiota profile at the same time. In this clinical trial, we evaluated the therapeutic efficacy of probiotics on HbA1c along with inflammatory cytokine levels of T1DM patients to determine an alternative administration mode for T1DM medication. The probiotics changed T1DM gut microbiota profile will be measured by next-generation sequencing (NGS). A randomized, double-blind, placebo-controlled trial was performed at China Medical University Hospital. T1DM patients between 6 and 18 years of age were enrolled. 27 patients were administered regular insulin therapy plus capsules containing probiotic strains subsp. AP-32, MH-68, and subsp. CP-9 daily for 6 months, and 29 patients were administered insulin therapy without extra probiotic supplement as placebo group. The variations of fasting blood glucose and HbA1c in these patients were analyzed. In addition, serum levels of inflammatory cytokines and anti-inflammatory cytokine were assessed using enzyme-linked immunosorbent assay. Patients' stool microbiota were all subjects to NGS analysis. NGS data showed elevated populations of and in the gut of patients with T1DM who were taking probiotics. Patients with T1DM who were administered probiotics showed significantly reduced fasting blood glucose levels compared with the before-intervention levels. The HbA1c levels of the patients also improved after administration of probiotics. The concentrations of IL-8, IL-17, MIP-1β, RANTES, and TNF-α were significantly reduced and were associated with an increased TGF-β1 expression after probiotic intervention. The persistence effect of glycemic control and immunomodulation were observed even 3 months after discontinuation of the probiotics. Here, we found that conventional insulin therapy plus probiotics supplementation attenuated T1DM symptoms than receiving insulin treatment only. Probiotics supplementation with insulin treatment changed gut microbiota and revealed better outcome in stabilizing glycemic levels and reducing HbA1c levels in patients with T1DM through beneficial regulation of immune cytokines. ClinicalTrials.gov, identifier NCT03880760.