Abstract Doxorubicin treatment was found to augment the expression of the extracellular matrix (ECM) protein fibulin-1 in cultured human breast cancer cell lines and in MDA-MB-361 tumors grown in athymic mice. Doxorubicin was also found to augment tumor expression of the fibulin-1–binding proteins fibronectin and laminin-1. Growth of breast cancer cell lines on Matrigel, an ECM extract containing fibulin-1 and laminin-1, resulted in lower levels of doxorubicin-induced apoptosis as compared with controls. Moreover, tumors formed by injection of athymic mice with MDA-MB-361 cells mixed with Matrigel were significantly more doxorubicin resistant and displayed lower levels of apoptosis compared with those that formed in the absence of Matrigel. Monoclonal antibodies against fibulin-1 reversed Matrigel-dependent doxorubicin resistance. Furthermore, small interfering RNA–mediated suppression of fibulin-1 expression in breast cancer cells resulted in a 10-fold increase in doxorubicin sensitivity as compared with control cells. Together, these findings point to a role for fibulin-1 in breast cancer chemoresistance. [Cancer Res 2007;67(9):4271–7]