Apatite is a common accessory mineral in iron oxide-apatite (IOA), magmatic Fe-Ti oxide, and metamorphosed sedimentary Fe deposits. Apatite chronology and oxygen isotopes in the IOA deposits have been extensively studied, however, such information in the metamorphosed sedimentary Fe deposits remains poorly understood. Here, we report detailed trace elements, oxygen isotopes, and U-Pb chronology of apatite from the Tianhu metamorphosed sedimentary Fe deposit in the eastern Tianshan, NW China. To our knowledge, this is the first report on the in situ oxygen isotopes of apatite from the metamorphosed sedimentary Fe deposits. Cathodoluminescence imaging of the apatite grains from the Tianhu massive Fe ores show a homogeneous texture, suggesting that they were formed by a single event rather than multiple superimposed events. These apatite grains contain abundant fluid inclusions, and show relatively high δ18O values of 7.9 ‰-14.0 ‰ and low Th and U contents, which are consistent with their metamorphic hydrothermal origin. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) dating of the studied apatite grains yielded a U-Pb age of 348.5 ± 18.5 Ma, indicating that the metamorphic enrichment of the Tianhu metamorphosed sedimentary Fe deposit occurred considerably later than the Ordovician ore-hosting rocks. Combined with previous studies, we suggest that the metamorphic massive ores of the Tianhu metamorphosed sedimentary Fe deposit formed in an orogenic setting. Our study highlights that a combination of trace elements, oxygen isotopes, and U-Pb chronology of apatite can be used as powerful tools to constrain the genesis of metamorphosed sedimentary Fe deposits.