Pituitary adenoma (PA) is one of the most common intracranial tumors, and owing to its special biological morphology and behavior, there is currently no effective treatment. miRNAs play crucial roles as diagnostic indicators and targets for the treatment of numerous cancer types. The objective of this research was to explore how miR-29a-3p influences the development of PA. We collected 25 pairs of PA tissue and normal pituitary tissue, followed by the subcutaneous injection of 5 × 107 HP75 cells into the left axilla of nude mice, creating a heterotopic PA xenograft tumor model for experimental study. TtT/GF and HP75 cell proliferation and tumor growth in nude mice were assessed using CCK-8, Transwell, and immunohistochemistry tests. Western blotting, RT‒qPCR and RIP were used to detect the expression and interaction of related proteins and genes. The expression of miR-29a-3p was upregulated in PA. Knockdown of miR-29a-3p can inhibit the proliferation, invasion and migration of TtT/GF and HP75 cells and reduce the epithelial mesenchymal transformation (EMT) of these cells. Furthermore, reducing miR-29a-3p levels suppressed the expression of Ki-67 in the PA tissues of nude mice and slowed tumor growth. From a mechanistic standpoint, miR-29a-3p can target COL6A6 and PTEN. Knockdown of miR-29a-3p inhibits the PI3K/Akt/CUX1 signaling pathway through simultaneously increasing COL6A6 and PTEN expression, thus inhibiting the proliferation, invasion, migration and EMT of PA cells and alleviating the progression of PA. Conversely, CUX1 can promote the expression of miR-29a-3p through a positive feedback loop and accelerate the development of PA. Our study suggests that downregulating the expression of miR-29a-3p may be a new target for the treatment of PA.© 2025. The Author(s).