Abstract: Objecitve To isolate and identify the pituitary adenoma stem-like cells from pituitary adenoma tissue. Methods Total RNA was prepared with 42 cases of pituitary adenoma tissue samples frozen in liquid nitrogen, the expression of Nestin was detected by RT-PCR. Tumor spheres were cultured in serum-free conditions and the immunefluorescence were used to detect the expression of stem cell markers such as Nestin, Sox2. Results The positive rate of Nestin mRNA expression was 88.1% (37/42). The cultured tumor spheres were found positive for Nestin, Sox2 by immunefluorescence detection. Conclusion Stem cell markers are expressed in the pituitary adenoma tissue and the pituitary adenoma stem-like cells can be cultured in serum-free condition.