Background: Prolactinomas are the most common secretory pituitary adenomas. The first line of treatment involves dopamine agonists (DAs); however, a subset of patients is resistant to such therapy. Recent studies suggest that dopamine can up-regulate TGF-beta 1 synthesis in rat pituitary lactotrophs whereas estradiol down-regulates TGF-beta 1. To date, the role of TGF-beta/Smad signaling in DAs-resistant prolactinomas has not been explored. Methods: High-content screening (HCS) techniques, qRT-PCR, Western blot, immunofluorescence and ELISA, were performed to determine the role of TGF-beta/Smad signaling in DAs-resistant prolactinomas. Results: We reported a significant down-regulation of TGF-beta/Smad signaling cascade in DAs-resistant prolactinomas compared to normal human anterior pituitaries. Following treatment with TGF-beta 1, the dopamine agonist, bromocriptine, and the estrogen antagonist (ER), fulvestrant in GH3 cells, we found that TGF-beta 1 and fulvestrant caused significant cytotoxicity in a dose- and time-dependent manner and activated Smad3 was detected following exposure to TGF-beta 1 and fulvestrant In addition, treating GH3 cells with fulvestrant increased active TGF-beta 1 levels and decreased PRL levels in a dose-dependent manner. Conclusion: TGF-beta/Smad signaling pathway may play an important role in DA-resistant prolactinomas and has the potential to be a viable target for the diagnosis and treatment of prolactinomas, particularly in patients who are resistant to DAs. (C) 2014 Elsevier Ireland Ltd. All rights reserved.