Altered expression of claudin-3 (CLDN3), a key cytoskeletal structural protein of the tight junctions in the epithelium, is associated with the development and metastasis of various human cancers. CLDN3 expression has been shown to be significantly associated with the prognosis of lung squamous cell carcinoma (SqCC). This study investigated the role of CLDN3 in inhibiting lung SqCC cell migration and invasion as well as the underlying molecular mechanisms. The CLDN3 levels were assessed between 20 paired lung SqCC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The ectopic CLDN3 overexpression or knockdown was generated by using a plasmid carrying CLDN3 cDNA or shRNA, respectively. CLDN3 expression was significantly reduced in lung SqCC tissues vs. the adjacent normal tissues. The ectopic CLDN3 overexpression markedly inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of lung cancer H520 cells, whereas CLDN3 knockdown had an inverse effect on SK-MES-1 cells. However, cell viability and plate colony formation assays showed that both CLDN3 knockdown and overexpression did not affect SqCC cell proliferation. Both tissue and cell data revealed that CLDN3 expression was significantly associated with the expression of the EMT biomarkers E-cadherin and Vimentin. Furthermore, CLDN3-modulated EMT and expression of the EMT markers were through regulation of the Wnt/beta-catenin signaling pathway. In conclusion, this study identified reduced CLDN3 expression in lung SqCC tissues, which was associated with the progression and metastasis of lung SqCC and was attributed to EMT by activation of the Wnt pathway. Thus, CLDN3 could be further evaluated as a novel biomarker for predicting the prognosis of lung SqCC and as a target for the treatment of lung SqCC in the future.