Estrogen is a risk factor for breast cancer. The isothiocyanate sulforaphane (SFN), found in cruciferous vegetables, has been identified as an effective chemopreventive agent, and may prevent or treat breast cancer by reversing estrogen-induced metabolic changes. Here, we investigated metabolic changes in estrogen receptor-positive breast cancer (MCF-7) cells treated with estradiol (E2) and/or SFN to identify key metabolite panels that might provide new insights into the underlying mechanisms of the antitumor effects of SFN. Gas chromatography-mass spectrometry and ultra performance liquid chromatography-mass spectrometry (UPLC-Orbitrap- MS) were used to obtain the metabolic profiles of MCF-7 cells. The data were analyzed using Student's t-test and multivariate statistics, including principal component analysis and partial least squares discriminant analysis. Hydroxymethylation was detected by UPLC-Orbitrap- MS and verified by immunofluorescence assay. We report that significant changes in metabolites induced by E2 and SFN were associated with differences in glycolysis and energy metabolism, and also amino acid, purine, and folic acid metabolism. E2 may alter methylation and hydroxymethylation status via the folic acid pathway. We also identified biomarkers that may be of use in interpretation of the metabolic pathways evoked by the effects of E2 and SFN on breast cancer cells. The identified biomarkers associated with metabolic pathways provide new insight into the chemopreventive mechanisms of SFN.