WW45 is a recently-discovered tumor suppressor gene. Overexpression of WW45 was found to significantly weaken proliferation and colony formation in a human breast cancer cell line, but the molecular mechanism of WW45's inhibitiory effect on proliferation was uncertain. It is a key transcription factor of the Hedgehog signaling pathway. In particular, the mechanism of Gli1's upstream proteins in regulating Gli1's nuclear import was not clear.We collected different breast cancer cell lines and detected WW45 and Gli1 expression by western blot. Gli1 expression was detected after WW45 was overexpressed in breast cancer cells. Gli1 and WW45 were transfected into breast cancer cells, and co-immunoprecipitation was used to detect whether the two proteins had physical interaction. We confirmed Gli1 blocks WW45-induced growth inhibition and colony formation in ZR-75-30 cells through cell functional experiments. Expression of WW45 negatively correlated with Gli1 expression in breast cancer cells. WW45 affected Gli1 intracellular localization though ww-PPxY/PsP interaction. Gli1 blocked WW45-induced growth inhibition and colony formation in ZR-75-30 cells. Our results strongly suggest that expression of WW45 negatively correlates with Gli1 expression in breast cancer cells. direct physical interaction occurred between WW45 and Gli1, and WW45 affected Gli1 intracellular localization though WW-PPxY/PsP interaction. Furthermore, Gli1 blocked WW45-induced breast cancer cell growth inhibition. AJTR Copyright © 2021.