We have previously found that the mRNA and protein levels of the folate receptor alpha (FRalpha) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FRalpha has not fully been determined. We investigated the effect of FRalpha over-expression in the mouse gonadotroph alphaT3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FRalpha were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FRalpha promotes cell proliferation. These effects were abrogated in the same alphaT3-1 cells when transfected with a mutant FRalpha cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FRalpha over-expressing cells. In summary, our data suggests that FRalpha regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.