BACKGROUND: Deeper studies on the pathological mechanism associated with invasiveness of non-functioning pituitary adenoma (NFPA) is imperative to find better treatments. This research was preliminarily conducted to investigate the correlation between the expression of Claudin-9 (CLDN9), Tyrosine kinase-2 (TYK2), Signal transducers and activators of transcription-3 (STAT3) and invasiveness in NFPA to illustrate the pathological mechanism. METHODS: Clinical data and surgical specimens of 12 patients with NFPA were collected and divided into invasive and non-invasive NFPA groups, comprising six patients for each group. CLDN9, TYK2 and STAT3 transcription and expression levels in the NFPA tissues of the two groups were detected by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and immunohistochemistry (IHC). The lentiviral plasmid transfection technique was used to develop a rat pituitary tumour GT1-1 cell line null control group (NC) and CLDN9-overexpressed experimental group (OE-CLDN9), and TYK2 and STAT3 transcription levels in the NC and OE-CLDN9 cell groups were detected using qRT-PCR. RESULTS: The CLDN9 and STAT3 expressions were significantly higher in invasive than in non-invasive NFPA tissues, whereas the TYK2 expression in invasive NFPA tissues was significantly lower than that in non-invasive NFPA (p < 0.001); The STAT3 upregulated (p < 0.001) and the TYK2 downregulated (p < 0.01) after the CLDN9 overexpression. CONCLUSION: Upregulated CLDN9 may increase the NFPA invasiveness through STAT3. In addition, low TYK2 expression might enhance the invasiveness in NFPA, which needs further studies to confirm. These results could provide a promising research leads for targeted treatment of NFPA.