Expression profile of microRNA (miRNA) in mouse oocytes and preimplantation embryos has been revealed by a novel high throughput microarray assay. A total of 97 (43 “new” and 54 known) including mouse, human, and predicted miRNAs have been discovered in the preimplantation mouse embryos which can be classified into developmental stage-dependent groups and non–stage-dependent group according to the statistical analysis of the expression patterns. Potential gene targets of each group of miRNAs are estimated by TargetsScan system and significantly changed signaling pathways and biological processes underlying these gene targets are searched by PANTHER classification system between the stage-dependent miRNAs and the non–stage-dependent miRNAs. Expression of some miRNAs is confirmed by reverse transcriptase-polymerase chain reaction. It is shown that dynamic synthesis and degradation of miRNAs coexists in the preimplantation development of mouse embryos. However, the overall quantity of miRNAs and percentage of the stage-dependent miRNAs increase as the preimplantation embryos develop. Developmental Dynamics 237:2315–2327, 2008. © 2008 Wiley-Liss, Inc.