In this study, we tested the hypothesis that overexpression of miR-9 and miR-29a may contribute to DPN development and progression. We performed a meta-analysis of miR expression profile studies in human diabetes mellitus (DM) and the data suggested that miR-9 and miR-29a were highly expressed in patients with DM, which was further verified in serum samples collected from 30 patients diagnosed as DM. Besides, ISL1 was confirmed to be a target gene of miR-9 and miR-29a. Lentivirus-mediated forced expression of insulin gene enhancer binding protein-1 (ISL1) activated the sonic hedgehog (SHH) signaling pathway, increased motor nerve conduction velocity and threshold of nociception, and modulated expression of neurotrophic factors in sciatic nerves in rats with DM developed by intraperitoneal injection of 0.45% streptozotocin, suggesting that ISL1 could delay DM progression and promote neural regeneration and repair after sciatic nerve damage. However, lentivirus-mediated forced expression of miR-9 or miR-29a exacerbated DM and antagonized the beneficial effect of ISL1 on DPN. Collectively, this study revealed potential roles of miR-9 and miR-29a as contributors to DPN development through the SHH signaling pathway by binding to ISL1. Additionally, the results provided an experimental basis for the targeted intervention treatment of miR-9 and miR-29a.