Background: Ischemia–reperfusion injury (IRI) is usually the key and often plays an irreversible role to induce flap compromise in microvascular tissue transfers. This article aims to profile the expression of micro-RNAs (miRs) in free flap surgeries following IRI. Methods: The miRs expression profiling was initially surveyed in rat epigastric flap vessels using Agilent 350-Microarrayed miRs after IRI, and then quantified by real-time reverse transcription polymerase chain reaction in flap vessels and tissues (n = 5) at three intervals: before induction of ischemia (normoxia without IRI, sham), 2 and 72 hours after reperfusion following 2 hours of ischemia. Furthermore, for seven patients with free anterolateral thigh flap reconstruction, the miRs expression patterns in these flaps before induction of ischemia (normoxia), at 2 and 72 hours after reperfusion following an ischemic interval were investigated. Results: Four miRs (miR-96, miR-193-3p, miR-210, and miR-21) of 350 tested rat miRs were found to be positively significant. In rat flap vessels, the upregulation of these miRs at 72-hour reperfusion was statistically significant. These patterns were not noted in rat flap tissues, except for miR-96. However, there seemed to be no significant difference in human flap vessels between normoxia and 2-hour reperfusion following ischemia. In human flap tissue, significant upregulation of miR-193-3p, miR-210, and miR-21 was detected at 72-hour perfusion. Conclusions: Our findings show some changes of four upregulated miRs in our model of IRI. We suggest that further investigation is needed to determine the role of miRs in IRI of microsurgical reconstruction. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012.