Four and a half LIM domain (FHL) protein 3 is a member of the FHL protein family that plays roles in the regulation of signal transduction, cell adhesion, survival, and mobility. FHL3 has been implicated in the development and progression of liver cancer. However, the biological function of FHL3 in other cancers remains unclear. Here, we show that FHL3 is downregulated in breast cancer patients. Using small interfering RNA (siRNA) knockdown and/or overexpression experiments, we demonstrated that FHL3 suppressed anchorage‐dependent and ‐independent growth of human breast cancer cells. The antiproliferative effects of FHL3 on breast cancer cell growth were associated with both the G1 and the G2/M cell cycle arrest, which was accompanied by a marked inhibition of the G1‐phase marker cyclin D1 and the G2/M‐phase marker cyclin B1 as well as the induction of the cyclin dependent kinase inhibitor p21 (WAF1/CIP1), a negative regulator of cell cycle progression at G1 and G2. These results suggest that FHL3 may play a role in the development and progression of breast cancer, and thereby may be a potential target for human breast cancer gene therapy. © 2011 IUBMB IUBMB Life, 63(9): 764–771, 2011