MicroRNAs are a class of endogenous noncoding small RNAs that have emerged as critical posttranscriptional regulators of the immune system. However, there have been few functional studies of the relationship between microRNA and immunosuppression. Here, the microRNA miR-125a-5p was identified as a key regulator of Tregs. We used miR-125a-5p knockout mice to explore the relationship between miR-125a-5p and Treg function and development in vivo and examine the underlying biochemical mechanisms. PCR and Western blotting were used to verify the regulatory relationship between miR-125a-5p and Foxp3 in mice in vivo, and flow cytometry was used to explore the proportions and numbers of Tregs in immune organs. The immunosuppressive effect of Tregs on other lymphocytes was analysed by measuring the proliferation of splenic lymphocytes after Con A induction. ELISA kits were used to detect inflammatory cytokines in mouse serum. In miR-125a-5p knockout mice, deletion of miR-125a-5p caused a decrease in Foxp3 expression, reduced Treg numbers, and an imbalance in inflammatory cytokines. Overall, our results indicated that miR-125a-5p contributes to Treg homeostasis and function, providing insights into potential miRNA-based therapies for autoimmune diseases.