Tumor-associated fibroblasts are the most abundant population in tumor stroma and impact on tumor initiation and progression. However, the biological function of tumor-associated fibroblasts in pituitary adenomas has not been fully elucidated to date. So, this study aims to clarify the function and significance of primary cultured pituitary adenoma-derived tumor-associated fibroblasts on rat pituitary adenoma cells. We identified primary cultured tumor-associated fibroblasts and normal fibroblasts based on the expression of α-smooth muscle actin as well as morphology. Furthermore, we investigated cell biological influences on rat pituitary adenoma cells through indirectly co-culturing tumor-associated fibroblasts with GH3 cells and subcutaneous xenograft model. All sorts of fibroblasts showed positive staining for α-smooth muscle actin. But α-smooth muscle actin and vascular endothelial growth factor highly expressed in invasive pituitary adenoma-derived tumor-associated fibroblasts compared to non-invasive pituitary adenoma-derived tumor-associated fibroblasts and normal fibroblasts. Besides, invasive pituitary adenoma-derived tumor-associated fibroblasts promoted the proliferation of GH3 cells in vitro as well as tumor growth in vivo. Finally, vascular endothelial growth factor was highly expressed in tumor specimens co-injected with invasive pituitary adenoma-derived tumor-associated fibroblasts. Our results suggested that invasive pituitary adenoma-derived tumor-associated fibroblasts displayed apparent growth promotion effects on rat pituitary cells both in vitro and in vivo accompanied by over-expression of vascular endothelial growth factor in invasive pituitary adenoma-derived tumor-associated fibroblasts and tumor specimens. ^* Lv, Zhang and Hu contributed equally in this manuscript as the co-first authors