The initiation of primordial follicle development is essential for female fertility, but the signals that trigger this process are poorly understood. Given the potentially important roles of microRNAs (miRNAs) in the ovary, we aimed to study the expression patterns and regulatory functions of miRNAs during the initiation of primordial follicle development. Expression patterns of miRNA in the neonatal mouse ovary were profiled by microarray, and 24 miRNAs whose abundances differed significantly between ovaries from 3- and 5-day-old mice were identified. Pathway enrichment analysis revealed that 48 signal transduction pathways are modulated by the up-regulated miRNAs and 29 pathways are modulated by the down-regulated miRNAs (P-value and false discovery rate < 0.001). A miRNA-mRNA regulatory network was established for TGF-beta signaling pathway-related genes. Among the miRNAs involved in this pathway, miR-145 was chosen for further analysis. Downregulation of miR-145 using an antagomir (AT) decreased the proportion and number of the primordial follicles and increased that of the growing follicles in the cultured ovaries (P < 0.05). The mean oocyte diameter in the primordial follicles was significantly greater in the AT group relative to the AT-negative control group (P < 0.05), whereas the mean oocyte diameter in growing follicles was smaller in the AT group than in the AT-negative control group. In addition, we confirmed that miR-145 targets Tgfbr2. The miR-145 AT caused an increase in TGFBR2 expression and activation of Smad signaling but did not affect the p38 MAPK or JNK pathway. These data suggest that miRNAs and the signaling pathways they modulate are involved in the initiation of primordial follicle development, and miR-145 targets Tgfbr2 to regulate the initiation of primordial follicle development and maintain primordial follicle quiescence.