Prototypical microRNAs (miRNAs) are 21 approximately 25-base-pair RNAs that regulate differentiation, carcinogenesis, and pluripotency by eliminating mRNAs or blocking their translation, in a process that is collectively termed RNA interference (RNAi). In zebrafish, RNAi mediated by miRNAs regulates early development, and in mice embryos that lack the miRNA precursor processor Dicer are nonviable. However, the roles of miRNAs in mammalian fertilization are unknown. In this report, we show using microarrays that miRNAs are present in mouse sperm structures that enter the oocyte at fertilization. The sperm contained a broad profile of miRNAs and a subset of potential mRNA targets, which were expressed in fertilizable metaphase II (mII) oocytes. Oocytes contained transcripts for the RNA-induced silencing complex (RISC) catalytic subunit, EIF2C3 (formerly AGO3). However, the levels of sperm-borne miRNA (measured by quantitative PCR) were low relative to those of unfertilized mII oocytes, and fertilization did not alter the mII oocyte miRNA repertoire that included the most abundant sperm-borne miRNAs. Coinjection of mII oocytes with sperm heads plus anti-miRNAs to suppress miRNA function did not perturb pronuclear activation or preimplantation development. In contrast, nuclear transfer by microinjection altered the miRNA profile of enucleated oocytes. These data suggest that sperm-borne prototypical miRNAs play a limited role, if any, in mammalian fertilization or early preimplantation development.