Polycystic ovary syndrome (PCOS) is a common endocrine abnormality in women characterized by a menstrual disturbance with chronic anovulation and hyperandrogenism, polycystic ovaries, and insulin resistance. MicroRNAs (miRNAs) are important fine-tune regulators involved in various physiological and pathological processes, but their actions are not fully understood. In this study, we observed the increased expression of miR27a-3p in the ovaries of mice with PCOS and explored its functions in primary mouse granulosa cells (mGCs) and the mouse granulosa-like tumor cell line, KK-1, using several approaches. QPCR results showed that miR-27a-3p expression was significantly higher in mGCs at the preantral follicle (PrF) stage. Using flow cytometry and hormone analysis, we found that overexpression of miR-27a-3p promoted apoptosis and inhibited estradiol (E-2) production in KK-1 cells. Moreover using a luciferase assay and Western blotting analysis, we verified that the gene of cyclic AMP response element (CRE)-binding protein 1 (Crebl) was a potential target of miR-27a-3p, which in effect hindered the expression of its downstream factor cytochrome P450 family 19 subfamily A polypeptide 1 (Cypl9a1). With the decrease of aromatase activity, testosterone (T) is reduced to dihydrotestosterone (DHT) and this exerts its effect of upregulation of the miR-27a-3p expression. The imbalance of androgen and E2 levels affected by miR-27a-3p and its function of promoting GC apoptosis could be involved in the pathophysiology of PCOS. Our results indicate that miR-27a-3p in PCOS GCs may play an important role in ovarian follicular development and provide new insights into GC dysfunction in PCOS.