Estradiol is a steroid hormone that not only plays an important role in ovarian follicular development but also is associated with many reproductive disorders. Owing to the importance of aromatase in the production of estradiol, the regulation of aromatase gene expression at the transcriptional level has been an extensive area of study for over two decades. However, its regulation at the posttranscriptional level has remained unclear. Here, we show that micro-RNA378 (miR-378) is spatiotemporally expressed in porcine granulosa cells, the cells that generate estradiol in the ovary during follicular development, in an inverse manner compared with the expression of aromatase. In vitro overexpression and inhibition experiments revealed that aromatase expression, and therefore estradiol production, by granulosa cells, is posttranscriptionally down-regulated by miR-378. Furthermore, site-directed mutation studies identified two binding sites in the 3'-untranslated region (3'-UTR) of the aromatase coding sequence that are critical for the action of miR-378. Interestingly, overexpression of the aromatase 3'-UTR enhanced aromatase expression at the protein level in granulosa cells, possibly mediated by the binding of miR-378 within this region, thereby reducing the binding of this micro-RNA to the endogenous aromatase 3'-UTR.