Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM‐ (MCF‐7, T47D, ZR‐75‐1), and ER‐/VIM‐ (MDA‐MB‐468, SK‐Br‐3) cell lines were uninvasive, while ER‐/VIM+ (BT549, MDA‐MB‐231, MDA‐MB‐435, MDA‐MB‐436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER‐/VIM+ cell lines were significantly more invasive than either the ER+/VIM‐ or ER‐/VIM‐ cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER‐/VIM+ cell lines MDA‐MB‐231, MDA‐MB‐435 and MDA‐MB‐436. Hs578T cells (ER‐/VIM +) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER‐/VIM+ MCF‐7ADR subline was significantly more active than the MCF‐7 cells in vitro, but resembled the wild‐type MCF‐7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF‐7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.