A phosphorylated prolactin is a kind of modified prolactin, which is produced through phosphorylation of native prolactin (PRL) by p21-activated kinase 2 (PAK2) in secretory granules in lactotrophs. Phosphorylated prolactin is involved in the regulation of the estrous cycle and in apoptosis in cancer cells, which seem to have important physiological and pathological roles, respectively. In previous research, it has been reported that estrogen induced the phosphorylation of prolactin in the mouse pituitary gland. However, the relationship between estrogen and PAK2 in the production of phosphorylated PRL has not been clarified yet. In order to examine whether PAK2 is involved in PRL phosphorylation by estrogen, we analyzed PAK2 protein levels in mice and phosphorylated prolactin levels in mouse pituitary cells by western blot analysis. The ratio of phosphorylated PAK2/total PAK2 was increased in estrogen implanted mice, but PAK2 protein and gene expression levels were decreased. In addition, the ratio of phosphorylated prolactin/non-phosphorylated prolactin was decreased in primary pituitary cells with introduced siPAK2. These findings suggest that estrogen could induce the phosphorylation of PRL through PAK2 activation. Therefore, this study contributes to better understanding of the mechanism of phosphorylated PRL production in physiological and pathological conditions associated with estrogen.