The promise of retinoids as chemopreventive agents in breast cancer is based on the differentiation and apoptosis induced upon their binding to the retinoic acid (RA) receptor beta (RARbeta). We have previously shown that HOXA5 induces apoptosis in breast cancer cells. In this study, we investigated whether RA/RARbeta and HOXA5 actions intersect to induce apoptosis and differentiation in breast cancer cells. We found that HOXA5 expression can be induced by RA only in RARbeta-positive breast cancer cells. We have, for the first time, identified the RA response element in HOXA5, which was found to be located in the 3' end of the gene. Chromatin immunoprecipitation assays showed that RARbeta binds directly to this region in vivo. Overexpression of RARbeta strongly enhances RA responsiveness, and knocking down RARbeta expression abolishes RA-mediated induction of HOXA5 expression in breast cancer cells. In addition, there is coordinated loss of both HOXA5 and RARbeta expression during neoplastic transformation and progression in the breast epithelial cell model, MCF10A. Knockdown of HOXA5 expression partially abrogates retinoid-induced apoptosis and promotes cell survival upon RA treatment. These results strongly suggest that HOXA5 acts directly downstream of RARbeta and may contribute to retinoid-induced anticancer and chemopreventive effects.