Background We have recently reported that RhoA may regulate the invasion and metastasis of breast cancer cells as an upstream signal of ezrin in vitro. In this study, we examined the relationship of RhoA signaling activity with ezrin expression in breast cancer and its prognostic significance in patients with breast cancer. Methods Paraffin tumor sections of breast cancer were collected retrospectively from 487 patients diagnosed between 2001 and 2004. Immunohistochemical methods were used to detect the expression of RhoA, phosphorylated (activated) RhoA, and ezrin. Results Ezrin overexpression was detectable in 15.2% of 487 invasive breast cancers. The majority (85.1%) of ezrin-overexpressing tumors coexpressed phosphorylated RhoA; 78.8% of tumors with phosphorylated RhoA cooverexpressed ezrin. Patients whose cancers showed overexpression of ezrin or expression of phosphorylated RhoA had shorter survival rates. Conclusions RhoA activation is important in human breast cancer due to its upregulation of ezrin; thus, agents that target phosphorylated RhoA may be useful in the treatment of tumors with ezrin overexpression.