Although the transforming growth factor-β (TGF-β) pathway has been implicated in breast cancer metastasis, its in vivo dynamics and temporal-spatial involvement in organ-specific metastasis have not been investigated. Here we engineered a xenograft model system with a conditional control of the TGF-β–SMAD signaling pathway and a dual-luciferase reporter system for tracing both metastatic burden and TGF-β signaling activity in vivo. Strong TGF-β signaling in osteolytic bone lesions is suppressed directly by genetic and pharmacological disruption of the TGF-β–SMAD pathway and indirectly by inhibition of osteoclast function with bisphosphonates. Notably, disruption of TGF-β signaling early in metastasis can substantially reduce metastasis burden but becomes less effective when bone lesions are well established. Our in vivo system for real-time manipulation and detection of TGF-β signaling provides a proof of principle for using similar strategies to analyze the in vivo dynamics of other metastasis-associated signaling pathways and will expedite the development and characterization of therapeutic agents.