Objective：To investigate the effect of knockdown of human epidermal growth factor receptor-3（HER3）expression on migration and invasion with irradiation（IR）in breast cancer cells and its molecular mechanism. Methods：Stable transfected breast cancer cells MCF-7 and ZR75-1 were established by short hairpin RNA（shRNA）to knockdown the expression of HER3 gene. The transfection efficiency was detected by Western blot. The control group transfected with empty virus and HER3 knockdown group were irradiated by 6-Gy X-ray. Cell healing assay and Transwell assay were used to analyze the migration and invasion ability of breast cancer cells. Western blot was used to detect the expression of Claudin-1. Results：After knockdown of HER3，HER3 protein expression in MCF-7 and ZR75-1 cells was significantly decreased. After IR，the migration and invasion increased，and the expression of Claudin-1 protein increased. However，knockdown of HER3 and combined IR reduced the migration and invasion of breast cancer cells，and decreased the expression of Claudin-1. Conclusion：Knockdown of HER3 reduces the migration and invasion of Luminal A breast cancer cells after IR. Its mechanism may be through the down-regulation of Claudin-1 expression.