Our study aims to elucidate the mechanisms how microRNA-129-5p (miR-129-5p) involved in neuroprotective effect of dexmedetomidine (DEX) on hypoxic-ischemic brain injury (HIBI) by targeting COL3A1 through the Wnt/β-catenin signaling pathway in neonatal rats. A total of 120 rats were obtained, among which 15 rats were selected as sham group and rest rats as model, DEX, DEX + negative control (DEX + NC), DEX + miR-129-5p mimics, DEX + miR-129-5p inhibitors, DEX + XAV-939 and DEX + miR-129-5p inhibitors + XAV-939 groups. Dual-luciferase reporter assay was performed for the target relationship between miR-129-5p and COL3A1. Weight rate and water content of cerebral hemisphere were detected. qRT-PCR and Western blotting were conducted to detect miR-129-5p expression and expressions of COL3A1, E-cadherin, T-cell factor (TCF)-4, and β-catenin. The DEX, DEX + miR-129-5p mimics, DEX + XAV-939 groups had increased weight rate of cerebral hemisphere, but decreased water content of left cerebral hemisphere, levels of COL3A1, β-catenin, TCF-4, and E-cadherin in hippocampus compared with the model and DEX + miR-129-5p inhibitors groups. COL3A1 was verified as the target gene of the miR-129-5p. Compared with the DEX + NC and DEX + miR-129-5p inhibitors + XAV-939 groups, the DEX + XAV-939 and DEX + miR-129-5p mimics groups had elevated weight rate of cerebral hemisphere, but reduced water content of left cerebral hemisphere, levels of COL3A1, β-catenin, TCF-4, and E-cadherin in hippocampus. Our findings demonstrate that miR-129-5p improves the neuroprotective role of DEX in HIBI by targeting COL3A1 through the Wnt/β-catenin signaling pathway in neonatal rats. This article is protected by copyright. All rights reserved.