The present study applied iTRAQ and LC-MS/MS techniques for proteome analysis and compared data between specimens of papillary thyroid microcarcinoma (PTMC) vs appropriate controls, in order to investigate the mechanisms underlying the invasion and metastasis process in PTMC development.Fresh-tissue specimens were collected from 40 patients with thyroid disease who underwent surgical treatment. Specimens were divided into four groups: normal histology (NH; n=8), benign thyroid tumor (BTT; n=10), classic PTMC with lymph node metastasis (PTC-LNM(+); n=11), and classic PTMC without lymph node metastasis (PTC-LNM(-); n=11). Proteomic studies were conducted on PTMC tissue samples without capsule invasion and with tumor diameter ranging from 0.5cm to 1cm, so as to focus the study on PTMC development excluding metastasis.A total of 8036 proteins were identified in the four groups. Based on protein function analysis, proteins that might be associated with PTMC invasion and metastasis were screened: alpha-actinin-1, alpha-1-antitrypsin, hepatoma-derived growth factor (HDGF), high-mobility group protein HMGI-C, and carbonic anhydrase 4. In addition, proteins involved in the focal adhesion pathway were examined. Immunohistochemistry confirmed the reliability of the iTRAQ results and the universality of differentially expressed proteins. The data showed that HDGF and high-mobility group protein HMGI-C are up-regulated in PTMC and that the focal adhesion pathway that promotes PTMC LNM is activated.These findings provide insight into the mechanisms underlying PTMC invasion and metastasis.