Aberrant microenvironments and loss of balance in cell-extracellular matrix signaling are associated with breast cancer invasion, metastasis, and resistance to therapy. We have recently shown that increased β₁ integrin signaling is involved in malignant progression and that inhibitory antibody to β₁ integrin leads to selective apoptosis and decreased proliferation in three-dimensional cultures and in xenograft models of breast cancer in vivo. To investigate the clinical importance of these findings, in the present study we examined the expression of β₁ integrin and extracellular β₁ integrin ligands fibronectin and laminin-1 in a cohort of 249 breast cancer patients who had a median follow-up of 8.4 years. Among the 149 scorable cases, the highest β₁ integrin intensity score (3+ versus 0–2+) was associated with significantly decreased 10-year overall survival of 48% versus 71% (P < 0.03) and decreased disease-free survival of 50% versus 80% (P < 0.05). Importantly, high fibronectin expression was associated with decreased overall and disease-free survival on univariate analysis (P < 0.04) and β₁ integrin intensity score was significantly correlated with fibronectin expression (Kendall's tau-b = 0.19; P = 0.03). In a multivariate Cox proportional hazards model, β₁ integrin intensity score remained a significant independent predictor of overall survival [hazard ratio (HR), 1.69; 95% confidence interval (95% CI), 1.19–2.38; P < 0.003] and disease-free survival (HR, 1.87; 95% CI, 1.21–2.88; P < 0.005). These findings show that β₁ integrin expression has potential prognostic value in invasive breast cancer and that coexpression of fibronectin may help identify patients with more aggressive tumors who may benefit from targeted therapy. [Cancer Res 2007;67(2):659–64]