MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs which can bind to completely or partially complementary sequences in the 3'UTR of target mRNAs, therefore degrading the mRNA or repressing translation. We previously reported that miR-378 played a role in estradiol production via suppression of aromatase translation in porcine granulosa cells and could affect oocyte maturation in vitro by inhibiting cumulus cell expansion. However, the role of miR-378 on ovary development in vivo is unknown. The current study aimed to uncover the molecular mechanism of miR-378 in regulating mouse follicular development via micro-injection of CMV-miR-378 lentivirus into the bursa of mouse ovary. The results showed that CMV-miR-378 lentivirus transduction in the mouse ovaries resulted in reduced ovary size, extended oestrous cycle (6-7 d in miR-378 overexpression group and 4-5 dyas in GFP control group) due to continuous oestrum, decreased percentage of oocytes in vitro maturation rate (IVM 60.8% vs. 89.4% in GFP control), increased apoptosis rate (Bax/Bcl2 in mRNA and protein level), decreased expression of genes associated with gap junction, such as connexin 43 (Cx-43) and connexin (Cx-37) and decreased expression of genes associated with follicular development, such as BMP15 and GDF9. Moreover, the number of pups/litter was consistently lower in the miR-378 group in each batch of the paired breeding. Our data suggest that miR-378 alters gene expression in cumulus cells and indirectly influences oocyte maturation competency, possibly via inhibition of oocyte-cumulus interaction or induction of apoptosis.