Cancer development involves the destruction of tight junctions, deprivation of cell polarity, and increased cell mobility. Claudin 16 (CLDN16) is a tight junction protein and plays important roles in the maintenance of cell polarity, cellular arrangement, adhesion, paracellular transport, and ionic permeability of various epithelia. A novel link protein, HAPLN3, functions in hyaluronic acid binding and cell adhesion. Both genes are hypothesized to be related to cancer development and metastasis. The purpose of this study was to estimate the roles of the genes CLDN16 and HAPLN3 in breast cancer. A total of 146 samples were collected from breast cancer tissues and their adjacent normal breast tissues. Reverse transcription and real-time polymerase chain reaction were used to estimate gene expression levels. There were significantly increased gene expression of CLDN16 (p<0.0001) and HAPLN3 (p<0.0001) among breast cancer tissues compared with normal tissues, irrespective of clinical pathological parameters. The absolute increased gene expression level of CLDN16 was significantly negatively correlated with estrogen (r=-0.46; p<0.0001) and progesterone receptor (r=-0.384; p=0.001) staining density. However, a significantly positive correlation (r=0.24; p=0.04) between the absolute increased HAPLN3 gene level and human epidermal receptor 2 staining density was found. There was no significant association between overall survival and the two gene expression levels. The gene up-expression of both CLDN16 and HAPLN3 was suggested to be involved in the development of breast cancer and to be a biomarker and target treatment for breast cancer.