Direct evidence for fault reactivation is crucial for understanding long-term fault behavior and reconstruction of fluid circulation and hydrocarbon migration and accumulation during active tectonics. However, absolute dating of fault reactivations and fluid circulation is still challenging due to dissolution and recrystallization of fault zone materials and obliteration of previous events by the latest deformation/hydrothermal event. The lower Ordovician limestone strata at 3.8 km depth in the Central Uplift of the Tarim Basin, northwest China experienced multi-stage regional tectonic activities that developed a series of NW–SE striking reverse faults and NE–SW oriented strike-slip faults. In this study, we performed in situ laser-ablation ICP-MS UPb dating and trace element characterization of calcites collected from borehole cores within one of the main fault zones in the Central Uplift of the Tarim Basin. A new in-house calcite standard AHX-1A (209.8 ± 1.3 Ma), collected from the Tarim Basin, calibrated against well-calibrated WC-1 and ASH-15 calcite standards, is used for mass-bias correction. We obtained in situ UPb ages of 9 calcite samples from two wells within a Paleozoic fault zone in the Tarim Basin. They are 456 ± 11 Ma, 454.7 ± 7.2 Ma, 450.4 ± 6.2 Ma, 435.2 ± 9.7 Ma, 328.0 ± 9.2 Ma and 307.6 ± 7.1 Ma from well TZ2, and 371 ± 18 Ma, 390.6 ± 6.0 Ma and 395 ± 14 Ma from well TZ4 (all reported uncertainties are 2σ). Combined with petrographic evidence, these results define at least six generations of secondary calcite precipitations in the fault zone, that occurred during 456 ± 11 to 450.4 ± 6.2 Ma (weighted mean 452.8 ± 4.3 Ma), 435.2 ± 9.7 Ma, 395 ± 14 to 390.6 ± 6.0 Ma (weighted mean 391.3 ± 5.5 Ma), 371 ± 18 Ma, 328.0 ± 9.2 Ma and 307.6 ± 7.1 Ma. Chondrite-normalized rare earth element (REE) + yttrium (Y) patterns of these calcite samples show prominent positive Gd and Y anomalies, lack of negative Ce anomaly, and enrichment in light REE but depletion in heavy REE, similar to the shallow marine platform host-rock limestone. The results suggest multi-stage fluid circulation under shallow burial, low temperature, and non-oxidizing environments along the fault zone, with the secondary calcite precipitating from a similar fluid source that originated from dissolution of host-rock carbonate or early-stage diagenesis. The detail petrographical and mineralogical analysis of this study, combined with the new interpretation of high-resolution seismic data, constrains two main episodes of faulting and related uplift during 456–435 Ma and 395–371 Ma, as well as a younger faulting episode with weaker intensity during 328–308 Ma. This study demonstrates that the central fault zone of the Tarim Basin experienced ~150 Ma of episodical reactivation during the Paleozoic, which has important implications for hydrocarbon exploration.