The objective of this study is to perform proteomic analysis of pleomorphic adenoma (PA) in the human parotid gland (PG) with comparison of normal PG. This is an individual prospective randomized controlled trial. This study was performed in a tertiary referral center. Tissue samples of PG and PA were taken after surgical excision of PG from 13 patients. Protein extracts were prepared and protein pools created from the soluble extracts were subjected to 2D-DIGE analysis. Proteins displaying regulation in their abundance were determined and identified using MALDIT-OF/TOF analysis. The identified proteins were subjected to STRING analysis for classification of the proteins based on their biological roles in metabolic pathways. Fifteen proteins, carbonic anhydrase 1, carbonic anhydrase 2, fibrinogen beta chain, alpha-amylase 1, heats hock protein hsp 90-alpha, clusterin, 78 kDa glucose-regulated protein, endoplasmin, alpha-amylase 2b, ATP synthase subunit alpha (mitochondrial), elongation factor 1-gamma, malate dehydrogenase, cytoplasmic, triosephosphate isomerase, receptor of activated protein c kinase 1, and aconitate hydratase, mitochondrial were down-regulated, whereas 11 proteins including ig kappa chain c region, serotransferrin, vimentin, annexin a5, glial fibrillary acidic protein, calreticulin, cartilage oligomeric matrix protein, microfibril-associated glycoprotein 4, 14-3-3 protein epsilon, fibulin-5, and f-box only protein 2 were up-regulated in PA samples in comparison to healthy parotid tissue. This study described the differences observed in protein expression patterns of the PA and normal PG. The results may provide new insights into the pathogenesis and diagnosis of PA in human PG.