The incomplete surgery section of invasive non-functional pituitary adenomas (NFPAs) carries the increased risks of complications and requires adjuvant radiotherapy and medications. It is necessary to clarify the molecular mechanisms and markers of invasiveness to guide the management of NFPA patients. The study aimed to proteomic variations of invasive and non-invasive NFPAs and sought the protein markers for invasive NFPAs. Invasive (n = 4) and non-invasive (n = 4) NFPA tissues were analyzed (n = 3-5/each tissue) with 2DE and PDQuest software. Twenty-four high-resolution 2DE gels were quantitatively compared to determine differentially expressed proteins (DEPs) between invasive and non-invasive NFPAs. Approximately 1200 protein spots were detected in each 2DE map, and 103 differential spots (64 upregulated and 39 downregulated) were identified. Among those 103 differential spots, 57 DEPs (30 upregulated and 27 downregulated) were characterized with peptide mass fingerprint and MS/MS. Gene-ontology (GO) and ingenuity pathway analyses of those DEPs revealed pathway networks including mitochondrial dysfunction, oxidative stress, mitogen-activated protein kinase signaling abnormality, TR/RXR activation, proteolysis abnormality, ketogenesis and ketolysis, cyclin-dependent kinase C signaling abnormality, and amyloid processing that were significantly associated with invasive characteristics of invasive NFPA. Those data demonstrate that proteomic variations exist between invasive and non-invasive NFPAs. 2DE-based comparative proteomics is an effective approach to identify proteomic variations and pathway network variations. Those findings will serve as a basis to understand the molecular mechanisms of invasive NFPAs and to discover protein markers to effectively manage patients with invasive NFPAs.