MicroRNAs (miRNAs) are present in serum and have the potential to serve as disease biomarkers. As such, it is important to explore the clinical value of miRNAs in serum as biomarkers for ischemic stroke (IS) and cast light on the pathogenesis of IS. In this study, we screened differentially expressed serum miRNAs from IS and normal people by miRNA microarray analysis, and validated the expression of candidate miRNAs using quantitative reverse-transcriptase polymerase chain reaction assays. Furthermore, we performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses to disclose functional enrichment of genes predicted to be regulated by the differentially expressed miRNAs. Notably, our results revealed that 115 miRNAs were differentially expressed in IS, among which miR-32-3p, miR-106-5p, and miR-532-5p were first found to be associated with IS. In addition, GO and KEGG pathway analyses showed that genes predicted to be regulated by differentially expressed miRNAs were significantly enriched in several related biological process and pathways, including axon guidance, glioma, MAPK signaling, mammalian target of rapamycin signaling, and ErbB-signaling pathway. In conclusion, we identified the changed expression pattern of miRNAs in IS. Serum miR-32-3p, miR-106-5p, miR-1246, and miR-532-5p may serve as potential diagnostic biomarkers for IS. Our results also demonstrate a novel role for miRNAs in the pathogenesis of IS.