In this study, we selected adult normal pituitary gland tissues from six patients during operations for pituitary microadenomas via the transsphenoidal approach for extended normal pituitary tissue resection around the tumor, and analyzed the protein expression of human normal pituitary using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry proteomics technology. The ten most highly expressed proteins in normal human pituitary were: alpha 3 type VI collagen isoform 5 precursor (abundance among tall pituitary proteins, 1.30%), fibrinogen beta chain preproprotein (0.99%), vimentin (0.73%), prolactin (0.69%), ATP synthase, H + transporting and mitochondrial F1 complex beta subunit precursor (0.52%), keratin Ⅰ (0.49%), growth hormone (0.45%), carbonic anhydrase Ⅰ (0.40%), heat shock protein 90 kDaⅠ (0.31%), and annexin V (0.30%). Based on the biological function classifications of these proteins, the top three categories by content were neuroendocrine proteins (abundance among all pituitary proteins, 40.1%), catalytic and metabolic proteins (28.3%), and cell signal transduction proteins (9.8%). Based on cell positioning classification, the top three categories were cell organelle (24.5%), membrane (20.8%), and cytoplasm (13.0%). Based on biological process classification, the top three categories of proteins are involved in physiological processes (42.9%), cellular processes (40.4%), and regulation of biological processes (9.1%). Our experimental findings indicate that a protein expression profile database of normal human pituitary can be precisely and efficiently established by proteomics technology. In this study, we selected adult normal pituitary gland tissues from six patients during operations for pituitary microadenomas via the transsphenoidal approach for extended normal pituitary tissue resection around the tumor, and analyzed the protein expression of human normal pituitary using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry proteomics technology. The ten most highly expressed proteins in normal human pituitary were: alpha 3 type VI collagen isoform 5 precursor (abundance among tall pituitary proteins, 1.30%), fibrinogen beta chain preproprotein , vimentin (0.73%), prolactin (0.69%), ATP synthase, H + transporting and mitochondrial F1 complex beta subunit precursor (0.52%), keratin Ⅰ (0.49% %), heat shock protein 90 kDa I (0.31%), and annexin V (0.30%). Based on the biological function classifications of these proteins, the top three categories by content w Based on cell positioning classification, the top three categories were cell organelle (24.5%), eu neuroendocrine proteins (abundance among all pituitary proteins, 40.1%), catalytic and metabolic proteins (28.3% , based on biological process classification, the top three categories of proteins are involved in physiological processes (42.9%), cellular processes (40.4%), and regulation of biological processes (9.1 %). Our experimental findings indicate that a protein expression profile database of normal human pituitary can be precisely and efficiently established by proteomics technology.