We aimed to identify putative H2O2-regulated miRNAs expressed in rice seedlings under oxidative stress by using a deep sequencing approach developed by Solexa (Illumina). Two small RNA libraries were constructed from H2O2-treated and control rice seedlings, and more than five million small RNA sequence reads were generated for each library. We identified seven H2O2-responsive miRNA families via expression profiling of the miRNAs based on a comparative miRNAomic analysis in combination with experimental validation. Furthermore, 32 miRNAs, 17 from known miRNA loci and 15 from novel miRNA loci, were also newly identified from the sequencing data. To the best of our knowledge, this is the first report of a systematic investigation of H2O2-regulated miRNAs and their targets in plants. Overall design: Examination of 2 different small RNA expression profilings in the control and H2O2 treated rice samples.