MicroRNAs (miRNAs) belong to a class of small non-coding RNAs that play important roles in complex biological processes through degradation of target mRNAs or repression of their translation. We exploited cross-species comparison to predict miRNAs and identified 266 genes encoding 274 mature miRNAs in Oryctolagus cuniculus. Comparative analyses among mammalian genomes demonstrated that most of the identified miRNAs and their clusters are ancient in origin and conserved among mammals but a few clades as well as some species-specific miRNAs exhibit an ongoing evolutionary process where gain and loss of individual miRNAs have occurred through tandem duplications and random mutations. Our microarray- and RT-PCR-based analyses and target prediction reveal specific expression patterns in brain, spleen, muscle, heart, and ovary, and significant over-representations in certain GO categories as regarded to their mRNA targets include genes that play key roles in signal transduction and transcriptional regulation.