Small and noncoding microRNAs (miRNAs) are known as key regulators of biological processes such as cell differentiation and tumor generation. They are also the important mediators of posttranscriptional gene silencing in both pathogenic and pathologic aspects of hypoxic-ischemic brain injury. miRNA microarray has been considered to be a high-throughput and precise analysis tool for detecting miRNA expression profiling, and it does greatly facilitate the research of the biological function of miRNAs. To investigate the changes of miRNA expression in cortex of neonatal rats with hypoxic-ischemic brain injury (HIBI) and the possible roles of miRNA in the pathogenesis of HIBI, we constructed the model of rat with HIBI and the cortex tissues were obtained 14 days after the HIBI operation. The large-scale miRNA microarrays and bio-informatics analysis were used to determine the differentially expressed miRNAs of HIBI rats compared with controls. Expression of 3 miRNAs (mir-429, mir-200b, and mir-182) was determined by quantitative real-time polymerase chain reaction. The results of miRNA expression profiles indicated that 5 miRNAs were up-regulated more than twice and 29 miRNAs were down-regulated more than twice compared with the normal control group. The results of the 3 miRNAs detected by quantitative real-time polymerase chain reaction were consistent with those detected by miRNA microarray. Hypoxic-ischemic brain injury rats have significant changes in miRNA expression, which demonstrated that miRNAs may play important roles in the pathogenesis of HIBI.