Survival factors are known to promote cell viability, and factor deprivation can be a potent apoptotic signal. Insulin-like growth factors are potent mitogens and inhibitors of apoptosis for many normal and neoplastic cells with insulin-like growth factor-I (IGF-I) being the most effective in many breast cancer cell lines. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues inhibit IGF-I-stimulated growth of MCF-7 human breast cancer cells. The aim of this study was to determine the relationship between inhibition of IGF-I responsiveness and induction of apoptosis by vitamin D analogues in breast cancer cells. Vitamin D analogues EB1089 and CB1093 inhibited autonomous and IGF-I-stimulated growth of MCF-7 and T47D cells and autonomous growth of IGF-I-insensitive Hs578T cells. In MCF-7 cells, IGF-I alone (4 nM) protected against apoptosis mediated by serum deprivation. Co-treatment with vitamin D analogues prevented the anti-apoptotic effects of IGF-I. In T47D cells, IGF-I treatment provided only partial protection against apoptosis induced by serum deprivation and co-incubation of serum-deprived cells with 100 nM CB1093 and IGF-I abrogated this partial protection. In Hs578T cells, addition of IGF-I did not prevent apoptosis induced by serum deprivation. However, treatment with CB1093 attenuated the protective effect of the serum in these cells. Our findings suggest that vitamin D analogues inhibit IGF-I signalling pathways to promote apoptosis in breast cancer cells.