AbstractSulforaphane (SFN) is a promising phytochemical with a wide range of antitumor activities. A comprehensive understanding of the effects of SFN on breast cancer based on the metabolome and microbiome is limited. Thus, we treated MCF‐7 cell‐transplanted nude mice with 50 mg/kg SFN. SFN inhibits breast cancer cell proliferation. SFN increased the levels of sulfate‐related metabolites and glutathione‐related metabolites and decreased tryptophan metabolites and methyl‐purine metabolites in urinary metabolic profile. SFN indirectly affected the activation of aryl hydrocarbon receptor by tryptophan metabolism. The ratio of SAM to methionine was decreased by SFN while the global DNA methylation was downregulated in tumor tissue. SFN decreased the sulfate‐reducing bacteriumDesulfovibrio, which is related to reduced methylation capacity, and increased the genusLactobacillusrelated to tryptophan metabolites with antitumor activities. In conclusion, we provide a perspective on the metabolome and microbiome to elucidate the antitumor activities of SFN.