In the present study, the efficacy of indole-3-carbinol (I3C), a key bioactive component of cruciferous vegetables, for prevention of cancer in offspring exposed in utero to the environmental carcinogen dibenzo[def,p]chrysene (DBC) was evaluated using an estrogen receptor β (ERβ) knockout mouse model. I3C was provided either through the maternal diet coincident with carcinogen exposure during pregnancy or directly to offspring postinitiation with DBC. I3C was effective at reducing T-cell acute lymphoblastic lymphoma/leukemia (T-ALL)-related mortality in offspring only if provided via the maternal diet, although a gender difference in the role of ERβ in mediating this response was evident. In female offspring, chemoprevention of T-ALL by maternal dietary I3C required expression of ERβ; survival in Esr2 wild-type and heterozygous female offspring was more than 90% compared with 66% in Esr2 null females. Alternatively, ERβ status did not significantly impact the transplacental chemoprevention by I3C in males. The possible role of ERβ in mediating lung carcinogenesis or chemoprevention by I3C was similarly complicated. Lung tumor incidence was unaltered by either dietary intervention, whereas lung tumor multiplicity was substantially reduced in Esr2 null females on the control diet and marginally lower in Esr2 null males exposed to I3C via the maternal diet compared with their wild-type and heterozygous counterparts. These findings suggest that I3C may act via ERβ to prevent or suppress DBC-initiated transplacental carcinogenesis but that the involvement of this receptor seems to differ depending on the cancer type and gender of the offspring.