Human clinically non-functioning pituitary adenomas (NFPAs) primarily cause headaches, visual impairment and hypopituitarism due to the effect of the mass of the tumor. Surgery is the first-line treatment for these tumors. To date, no efficacious medical therapy exists for non-functioning adenomas. Previous studies have demonstrated that the Notch3 receptor is involved in the pathogenesis of various types of malignancies, including human NFPAs. The current study focused on the expression of the Notch3 receptor and its ligand Jagged1 in three types of pituitary adenomas and in the normal pituitary gland. Using quantitative real-time RT-PCR assays and western blot analyses, upregulated Notch3 and Jagged1 were observed in human NFPAs, but not in normal human pituitary glands or in hormone-secreting adenomas. Furthermore, Notch3 was positively correlated with Jagged1 at the mRNA and protein levels. These data indicate that Notch3 and Jagged1 may play an important role in the initiation and proliferation of human non-functioning adenomas, and there may be an interaction between Notch3 and Jagged1 in this process. Our study further elucidates the role of the Notch3 signaling pathway in the tumorigenesis of human NFPAs and provides a potential therapeutic target for the medical treatment of these tumors.