Background The specific underlying pathogenesis of prolactinoma has not been clarified yet, to the best of our knowledge. p38 mitogen-activated protein kinase (MAPK) signaling including p38 alpha MAPK (MAPK14), p38 beta (MAPK11), p38 gamma (MAPK12) and p38 delta (MAPK13) is associated with the development and progression of several types of cancer. Methods Immunofluorescence analysis was performed on the prolactin (PRL) and MAPK14 expressions of pituitary gland in C57BL/6 mice and human prolactinoma specimen. In the present study, the role of MAPK14 in prolactinoma was determined using estradiol-induced mice and dopamine D2 receptor knockout (DRD2(-/-)) mice models in C57BL/6 wild-type (WT), MAPK14(-/-)and DRD2(-/-)MAPK14(+/-)mice. GH3 cells were transfected with different sets of MAPK14 small interfering RNA, which to study MAPK14 and PRL expression in GH3 cells. Results Immunofluorescence analysis showed that PRL and MAPK14 expression were colocalized and increased in the pituitary gland of mice and human prolactinoma specimen compared with the control specimen. It was shown that PRL and MAPK14 expression was colocalized and increased significantly in the pituitary gland of estradiol-injected prolactinoma mice compared with the control mice. Knockout of MAPK14 significantly inhibited tumor overgrowth, and PRL expression was decreased in estradiol-induced mice. Furthermore, MAPK14 knockout of DRD2(-/-)MAPK14(+/-)mice significantly reduced the overgrowth of pituitary gland and PRL production and secretion compared with DRD2(-/-)mice. MAPK14 knockout using siRNA inhibited PRL production in GH3 cells. Conclusion These results suggest that MAPK14 serves a promoting role in the formation of prolactinoma, and highlights the potential of MAPK14 as a potential therapeutic target in the treatment of prolactinoma.