1. The Tibetan chicken, which is an indigenous breed living on the Tibetan Plateau, exhibits hypoxic adaptations to its high-altitude environment. However, the molecular mechanism behind this hypoxic adaptation is still unclear. This study aimed to investigate differentially expressed miRNAs involved in hypoxic adaptation through high-throughput RNA sequencing. 2. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the differentially expressed miRNAs and their target genes in chicken embryonic heart tissues and fibroblasts. Luciferase reporter assays were performed to confirm the relationship between miRNAs and target genes. 3. The study identified 37 differentially expressed miRNAs in Tibetan chicken embryonic heart tissues, including 20 up- and 17 down-regulated miRNAs, compared to those found in lowland chickens. Differentially expressed miRNAs were mainly involved in biological processes, such as cell cycle arrest, toll-like receptor signalling pathways, and I-kappa B kinase/NF-kappa B signalling. The data showed that gga-miR-34 c-5p was significantly upregulated in Tibetan chicken tissues and hypoxic fibroblasts, while EHHADH, a target gene of gga-miR-34 c-5p, was downregulated. Moreover, gga-miR-34 c-5p dramatically decreased the luciferase activity of the wild EHHADH, whereas no effect on the mutational EHHADH was found. 4. This study identified miRNA expression profiles in the Tibetan chicken and suggested that miR-34 c-5p acts as a novel miRNA associated with hypoxic adaptation. This facilitates the understanding of molecular mechanisms that underlie long-term exposure to hypoxia.