Abstract: The miRNA cluster mir-17-92 is conserved in vertebrates and plays crucial roles in cell proliferation, differentiation, apoptosis and animal development. The mir-17-92 cluster also acts as an oncogene that is expressed in variety of cancers. Despite extensive study, the molecular mechanism underlying its functions is not fully understood. The fact that miRNAs in the same cluster are functionally related was used in the present study to investigate the function, and regulation of the chicken mir-17-92 cluster with GO analysis, pathway analysis, and binding site distribution analysis. The investigation found that the chicken mir-17-92 cluster regulated several vital cellular signaling pathways, including the MAPK, Wnt and TGF-β signaling pathway. A miRNA binding site distribution analysis found that multiple miRNAs within the mir-17-92 cluster targeted the same genes, suggesting that the miRNA members of the mir-17-92 cluster act synergetically to regulate target genes. This study paves the way for future investigation into how the mir-17-92 cluster may regulate key cellular processes involved in cancer and development.