We have previously reported the synthesis of VP-128, a new 17β-oestradiol (E2)-linked platinum(II) hybrid with high affinity for oestrogen receptor α (ERα). In the present study, we have investigated the anti-tumour activity of VP-128 towards breast cancer cells in vitro and in vivo. We used human ERα-positive (MCF-7) and -negative (MDA-MB-468) cells as a model for treatment with increasing doses of VP-128, cisplatin or E2 in vitro and for xenograft experiments in nude mice in vivo. Compared with cisplatin, VP-128 showed markedly improved in vitro and in vivo anti-tumour activity towards ERα-positive MCF-7 breast cancer cells, without increased systemic toxicity. In these caspase-3-deficient cells, treatment with VP-128 overcame weak cellular sensitivity to cisplatin in vitro and in vivo. In these cells, only the hybrid induced apoptosis in an ERα-dependent manner, inactivated both X-linked inhibitor of apoptosis protein and Akt, and induced selective nuclear accumulation of ERα and the expression of ER-regulated genes c- myc and tff1, which was blocked by ERα-specific antagonist ICI 282 780. In the case of ERα-negative MDA-MB-468 cells, VP-128, but not cisplatin, induced nuclear accumulation of apoptosis-inducing factor and inhibited c- myc expression. However, VP-128 did not show enhanced in vivo anti-tumour activity compared with cisplatin. These results reveal two different modes of action for VP-128 in ERα-positive and -negative breast cancer cells, and highlight the promising therapeutic value of this unique E2-platinum hybrid for selective targeting of hormone-dependent cancers.