The formation of ovarian follicles and subsequent development after birth are critical processes for female reproduction, and inappropriate coordination of these processes contributes to ovarian pathologies, such as premature ovarian failure and infertility. Identification and functional investigation of the factors involved in follicular assembly and the initial recruitment will be of great significance to the understanding of the female reproduction process. In this study, we examined the roles of transcription factor heterogeneous nuclear ribonucleoprotein K (Hnrnpk) in rat primordial folliculogenesis using RNA interference knockdown strategies. Reducing Hnrnpk mRNA levels via Hnrnpk small interfering RNAs to neonatal ovaries resulted in a substantial loss of naked oocytes, primordial and primary follicles. Structure disorganization of the ovary characterized by groups of oocytes arranged in nests, clusters of somatic cells not associated with any oocytes and many highly condensed oocyte nuclei was observed. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay demonstrated that these abnormalities may be partially attributable to abnormal apoptosis of oocytes. Furthermore, the microarray analysis showed that 63 genes changed significantly (≥2-folds or ≤0.5-fold) between the ovaries treated with Hnrnpk small interfering RNAs and the controls, with 22 up-regulated genes and 41 down-regulated genes. These differentially expressed genes were involved in several critical biological processes in ovarian development. These results suggest that transcription factor Hnrnpk is a key regulator for primordial follicle assembly and development, which provides a new potential therapeutic target to regulate ovarian function and treat ovarian disease.