Plurihormonal pituitary adenomas (PHPAs) are defined as those pituitary adenomas secreting two or more hormones that differ in chemical composition, immunoreactivity, and biologic effects. Since the pathogenesis of these adenomas is not well understood, our study aimed to explore mechanisms underlying the pathogenesis of PHPAs. We used bead-based fiber-optic arrays (Illumina Human GeneChip WG-6 v3.0) to examine the gene expression profiles in seven PHPAs compared with three normal pituitary glands. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse-transcription polymerase chain reaction. We then performed pathway analysis of all differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Our array analysis showed significant increases in the expression of 6 genes and decreases in 334 genes and 15 expressed sequence tags in the PHPAs. Bioinformatic analysis showed that genes HIGD1B, EPS8, ECT2, and BTG2 might play an important role in the tumorigenesis and progression of PHPAs. Pathway analysis showed that the p53 and Notch signaling pathways may play an important role in tumorigenesis and progression of PHPAs, and extracellular matrix (ECM)-receptor interactions likely play a role in the inhibition of invasion and metastasis in these tumors. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of PHPAs. Bead-based fiber-optic arrays combined with pathway analysis of gene expression data appears to be a valid method for investigating the pathogenesis of tumors.