Estrogen is a steroid hormone that has been implicated in a variety of cellular and physiological processes and in the development of diseases such as cancer. Here we show a remarkable widespread microRNA (miRNA) downregulation in the zebrafish (Danio rerio) liver following 17β-estradiol (E2) treatment. This unique miRNA expression signature in the fish liver was further supported by a combination of computational predictions with gene expression microarray data, showing a significant bias toward upregulation of miRNA target genes after E2 treatment. Using pathway analysis of target genes, their involvement in the processes of cell cycle, DNA replication, and proteasome was observed, suggesting that miRNAs are incorporated into robust regulatory networks controlled by estrogen. In oviparous vertebrates, including fish, the formation of yolky eggs during a process known as vitellogenesis is regulated by estrogen. Microarrays were used to compare miRNA expression profiles between the livers of vitellogenic and nonvitellogenic zebrafish females. Among the upregulated miRNAs in vitellogenic females, were five members of the miR-17-92, a polycistronic miRNA cluster with a role in cell proliferation and cancer. Furthermore, a number of miRNA target genes related to fish vitellogenesis were revealed, including vtg3, a putative target of miR-122; the most abundant miRNA in the liver. Moreover, several of the differentially expressed miRNAs were only conserved in oviparous animals, which suggest an additional novel level of regulation during vitellogenesis by miRNAs and consequently, improves our knowledge of the process of oocyte growth in egg-laying animals.