Aims Fructooligosaccharides (FOSs) known for their health properties and beta-(2 -> 6)-levan-type FOSs have shown prebiotic and immunomodulatory activities that overcome those of commercial beta-(2 -> 1)-FOSs, but costs do not favour their use. Moreover, FOSs can reach the bloodstream through the diet, and little is known about their direct effect on cells. The aim of this work was to produce high-content FOSs by Bacillus subtilis natto CCT7712 in a bioreactor using commercial sucrose and to evaluate their antiproliferative effects in OVCAR-3 cells. Methods and Results FOS production reached 173 center dot 60 g l(-1), 0 center dot 2 vvm aeration and uncontrolled pH. Levan-type FOSs, composed of beta-(2 -> 6) links and mainly GF(3) (6-nystose), were identified using RMN spectroscopy, FT-IR and ESI-MS. FOSs decreased the viability and proliferation of OVCAR-3 cells, and the effects were associated with an increased pro-inflammatory response by the induction of IL-8 and TNF-alpha, and the repression of ER-beta genes. The metabolic profiles showed disruption of cellular homeostasis that can be associated with a decrease in proliferation. Conclusions The high production of levan-type FOSs from B. subtilis natto CCT7712 in a bioreactor was achieved, and they showed antiproliferative potential in OVCAR-3 cells. Significance and Impact of the Study FOS could be a good target for future therapeutic studies and commercial use.