Inositol hexaphosphate (InsP6 or IP6) is an active ingredient of high fiber diet that has anti-cancer action in both in vitro and in vivo models. Recently we have demonstrated that InsP6 significantly inhibits DMBA-induced rat mammary cancer in vivo. To test the hypothesis that InsP6 mediates its function via inhibition of cell proliferation irrespective of hormonal dependence, its effect on growth inhibition and differentiation were studied in two human mammary carcinoma cell lines with different estrogen receptor status. Cell growth was measured by MTT incorporation assay, DNA synthesis by 3H-Tdr uptake and differentiation marker lactalbumin by immunocytochemistry. Dose-dependent growth inhibition was observed in both estrogen receptor-positive (MCF-7) and receptor-negative cells (MDA-MB-231). Statistically significant growth inhibition (p < 0.05) was observed starting at 1 mM InsP6 as early as after the first day of treatment and continued up to 6 days for both the cell lines. DNA synthesis in both the cell lines was suppressed by InsP6 occurring as early as 3 h after the beginning of treatment and continued up to 48 h; significant inhibition (p < 0.05) started at 1 mM InsP6 after 6 h of treatment. Compared to untreated cells, a 5-fold (p < 0.05) and 22-fold (p < 0.01) increase in expression of lactalbumin, associated with luminal cell differentiation was identified by immunocytochemistry after 48 h of treatment with 1 and 5 mM InsP6. Our data show that the inhibition of DNA synthesis and cell growth and induction of differentiation of human mammary cancer cell lines by InsP6 is independent of the estrogen receptor status of the cells. Taken together with results from in vivo studies, InsP6 may be an important candidate for the prevention and treatment of human breast cancer.