Purpose: To evaluate the effect of microRNA-489 (miR-489) on pituitary prolactinoma and its mechanisms of action. Methods: MMQ and GH3 cells were transfected with miR-489, cell viability assessed with cell counting kit-8 (CCK-8), and clone spots was evaluated by colony formation assay. Transwell assay was applied to measure cell migration and invasion while TargetScan was employed to the presumed targets of miR-489, followed by luciferase reporter assays. was MiR-489 and p21-activated kinase 3 (PAK3) gene expression were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR. Protein levels of PAK3 were measured using western blots. Results: Transfection significantly increased miRNA-489 levels (p < 0.01). Cell viability, number of clone spots, as well as cell migration and invasion diminished in MMQ and GH3 cells following miR-489 transfection when compared to miR-NC mimic group (p < 0.01). The presumed binding site of miRNA- 489 was located in 3′-untranslated region (UTR) of PAK3, and miR-489 transfection repressed luciferase activity with the wild-type 3′-UTR (p < 0.05). In addition, miR-489 decreased PAK3 levels in MMQ and GH3 cells. Knockdown of PAK3 significantly suppressed cell viability, clone formation ability, as well as cell migration and invasion when compared to negative control (p < 0.01). Conclusion: MiR-489 overexpression suppresses pituitary prolactinoma by targeting PAK3, thus providing a potential therapeutic strategy for the management of pituitary prolactinoma.