MicroRNAs play important regulatory roles in many physiological processes. This study investigated potential involvement of microRNAs in hepatic ischemia/reperfusion injury and ischemic preconditioning in mice. MicroRNAs with significant changes in expression in the livers upon ischemic preconditioning following ischemia/reperfusion injury were detected by microRNA microarrays. Seventy-eight microRNAs (40 down/38 up) exhibiting more than twofold differences were identified in the livers upon ischemia/reperfusion injury. Among these microRNAs, four microRNAs were further significantly downregulated by ischemic preconditioning in comparison to nonpreconditioned controls. These included mmu-miR-23a, mmu-miR-326, mmu-miR-346_MM1, and mmu-miR-370, all of which were positively correlated with the severity of ischemic injury. The expression of mmu-miR-326 was further confirmed by quantitative real-time RT-PCR, and CCAAT/enhancer binding protein alpha was predicted by computer-aided algorithms to be a downstream target of this microRNA. In summary, our study showed a distinctive miRNA expression pattern in mouse livers in response to ischemic preconditioning following ischemia/reperfusion injury. Further studies on the miRNAs identified herein may enhance the understanding of miRNA-based mechanisms of hepatic ischemia/reperfusion injury and ischemic preconditioning.