Calcium signaling is a critical regulator of cell proliferation. Elevated expression of calcium channels and pumps is a characteristic of some cancers, including breast cancer. We show that the plasma membrane calcium channel TRPV6, which is highly selective for Ca(2+), is overexpressed in some breast cancer cell lines. Silencing of TRPV6 expression in a breast cancer cell line with increased endogenous TRPV6 expression leads to a reduction in basal calcium influx and cellular proliferation associated with a reduction in DNA synthesis. TRPV6 gene amplification was identified as one mechanism of TRPV6 overexpression in a subset of breast cancer cell lines and breast tumor samples. Analysis of two independent microarray expression datasets from breast tumor samples showed that increased TRPV6 expression is a feature of estrogen receptor (ER)-negative breast tumors encompassing the basal-like molecular subtype, as well as HER2-positive tumors. Breast cancer patients with high TRPV6 levels had decreased survival compared with patients with low or intermediate TRPV6 expression. Our findings suggest that inhibitors of TRPV6 may offer a novel therapeutic strategy for the treatment of ER-negative breast cancers.