Clinical and animal studies have shown that coexpression of the receptor tyrosine kinases HER2 and epidermal growth factor (EGF) receptor (EGFR) indicates a highly metastatic phenotype of breast cancer. In a cellular model of this phenotype using differential gene expression analysis, we identified TOB1 to be up-regulated depending on EGF stimulation and transduction through phosphorylation of HER2 tyrosine 1248. mRNA expression analysis of breast cancers from a cohort of node-negative patients showed significantly shortened distant metastasis-free survival for patients with high TOB1 expression. In subsequent tissue microarray studies of 725 clinical samples, high HER2 and EGF protein levels were significantly correlated with TOB1 expression in breast cancer, whereas EGFR and EGF levels correlated with TOB1 phosphorylation. We did not observe a correlation between TOB1 expression and cyclin D1, which was previously suggested to mediate the antiproliferative effect of unphosphorylated TOB1. A positive correlation of TOB1 phosphorylation status with proliferation marker Ki67 suggests that elevated TOB1 phosphorylation might abrogate the antiproliferative effect of TOB1 in breast cancer. This suggests a new regulatory role for TOB1 in cancer progression with particular significance in HER2- and/or EGFR-positive breast cancers.