Disruption or loss of tight junction structure and function is associated with tumor growth, invasion and metastasis in tumors of human epithelial origin. Since claudin is the most important backbone protein of tight junctions, the downregulation or loss of claudin expression is hypothesized to be important for tumor development and metastasis. In the current study, RNA interference (RNAi) was used to knock down the expression of claudin‑6 to investigate the effect of claudin‑6 downregulation on the malignant phenotype in the human breast epithelium cell line HBL‑100. The junctional function was investigated by measuring the transepithelial electrical resistance across the confluent epithelial cell layer. Manual cell counting and wound healing assays were performed to examine cell proliferation and migration. Changes in matrix metalloproteinase‑2 (MMP‑2) expression and activity were examined by reverse transcription polymerase chain reaction (RT‑PCR) and gelatin zymography. The expression of p38 mitogen‑activated protein kinases (MAPKs) and phosphorylated p38 MAPK were measured by western blot analysis. Claudin‑6 knockdown resulted in significantly lower transepithelial electrical resistance (P<0.001), higher growth rate (P<0.001) and migratory ability (P<0.001) accompanied with an increased MMP‑2 expression and activity (P<0.001). Furthermore, a decreased expression of phosphorylated p38 MAPK (P<0.001) was detected in HBL‑100 cells. These observations support the hypothesis that a decreased expression of claudin‑6 contributes to the malignant progression of human breast cancer.