A lithogeochemical, mineral chemical, isotopic, and fluid inclusion study of barren, low-, and high-grade Au-mineralized samples from the shear zone-hosted Amantaytau gold deposit, Uzbekistan, shows that the local host rocks, Late Ordovician–Earlz Silurian carbonacous shales, are likely to have been an important source of Au, As, Ni, and S in the formation of the deposit. Syn-depositional pyrite in these shales contains on average 0.23 ppm Au, 1,083 ppm As, and 861 ppm Ni. The distribution of rare earth elements (REE) indicates a homogeneous source of light REE, whereas the heavy REE distribution reflects most likely primary variations in the sediments. The mineralized zone is marked by a positive Eu anomaly, which supports reducing conditions during the mineralization. A hydrothermal overprint by an aqueous–carbonic fluid is reflected in a high-grade Au-mineralized sample by δ13C values of −13.0 ‰ (V-PDB). The δ34S values in pyrite (−0.13 to +7.30 ‰ CDT) from barren and mineralized samples are consistent with marine sulfate being the principal source of the ore sulfur. Assuming a formation temperature of between 300 and 400 °C for the main stage of mineralization, as indicated by the alteration mineral assemblage, the calculated δ18Ofluid is between 9.5 and 13.4 ‰ V-SMOW, which points at a metamorphic origin of the ore fluid.