Melanoma differentiation‐associated gene‐7/interleukin‐24 (mda‐7/IL‐24) displays a broad range of antitumor properties including cancer‐specific induction of apoptosis, inhibition of tumor angiogenesis and modulation of antitumor immune responses. In our study, we elucidated the role of MDA‐7/IL‐24 in inhibiting growth of breast cancer‐initiating/stem cells. Ad.mda‐7 infection decreased proliferation of breast cancer‐initiating/stem cells without affecting normal breast stem cells. Ad.mda‐7 induced apoptosis and endoplasmic reticulum stress in breast cancer‐initiating/stem cells similar to unsorted breast cancer cells and inhibited the self‐renewal property of breast cancer‐initiating/stem cells by suppressing Wnt/β‐catenin signaling. Prevention of inhibition of Wnt signaling by LiCl increased cell survival upon Ad.mda‐7 treatment, suggesting that Wnt signaling inhibition might play a key role in MDA‐7/IL‐24‐mediated death of breast cancer‐initiating/stem cells. In a nude mouse subcutaneous xenograft model, Ad.mda‐7 injection profoundly inhibited growth of tumors generated from breast cancer‐initiating/stem cells and also exerted a potent “bystander” activity inhibiting growth of distant uninjected tumors. Further studies revealed that tumor growth inhibition by Ad.mda‐7 was associated with a decrease in proliferation and angiogenesis, two intrinsic features of MDA‐7/IL‐24, and a reduction in vivo in the percentage of breast cancer‐initiating/stem cells. Our findings demonstrate that MDA‐7/IL‐24 is not only nontoxic to normal cells and normal stem cells but also can kill both unsorted cancer cells and enriched populations of cancer‐initiating/stem cells, providing further documentation that MDA‐7/IL‐24 might be a safe and effective way to eradicate cancers and also potentially establish disease‐free survival.