The Nanmingshui gold deposit, located in the eastern segment of the Kalamaili gold belt (KGB), is hosted by the sub-greenschist facies rocks of the Lower Carboniferous Jiangbasitao Formation. The genesis of this deposit, however, has been debated for decades because of controversial constraints on the P-T-X conditions and origins of hydrothermal fluid and mineralization age. In this study, we present gold-bearing sulfide compositions, fluid inclusions, H-O isotopes, and the results of hydrothermal zircon U-Pb dating to provide new insights into the genesis of the gold deposit. Three gold mineralization stages are recognized: quartz-pyrite-minor native gold veins (early), quartz-tourmaline-arsenopyrite-pyrite-gold-polymetallic sulfide veins (middle), and quartz-calcite veinlets (late). Gold predominantly occurs as native gold with high fineness ranging from 941 to 944 in sulfides and quartz, and some as solid solutions (Au+) within the lattice of pyrite and arsenopyrite. Three types of primary fluid inclusions are identified in hydrothermal quartz: CO2-H2O (C-type), aqueous (W-type), and pure CO2 (PC-type) inclusions. The early-stage quartz mainly contains C-type and minor W-type inclusions, with total homogenization temperatures (Th) of 220-339 degrees C, salinities of 0.4-3.7 wt.% NaCl eqv., and bulk densities of 0.66-1.01 g/cm3. All three types of inclusions are observed in the middle-stage quartz, of which the C- and W-type inclusions yield Th values of 190-361 degrees C, with salinities of 0.4-6.0 wt.% NaCl eqv. and bulk densities of 0.69-0.99 g/cm3. The late-stage quartz contains only W-type inclusions that have lower Th values of 172-287 degrees C, higher salinities of 1.4-6.9 wt.% NaCl eqv., and bulk densities of 0.79-0.95 g/cm3. Trapping pressures estimated from C-type inclusions in the early and middle stages cluster at 280-340 MPa and 220-310 MPa, respectively, corresponding to metallogenic depths of 10-13 km and 8-11 km. The H-O isotopic compositions (delta 18Owater = 1.8-10.9 parts per thousand, delta D = -99 to -62.9 parts per thousand) and microthermometric data indicate that the ore-forming fluids belong to medium-high-temperature, low-salinity, medium-density, and CO2-rich-H2O-NaCl +/- CH4 +/- N2 systems, probably originating from metamorphic water. Fluid immiscibility is a crucial mechanism for gold precipitation. Additionally, the U-Pb dating of hydrothermal zircons, from the auriferous quartz-tourmaline vein, yield a weighted mean 206Pb/238U age of 314.6 +/- 9.6 Ma. Taking all of the above, the Nanmingshui deposit can be reasonably classed as a typical mesozonal orogenic gold deposit in the KGB, which was formed in a Late Carboniferous tectonic transition from syn-collision between the Jiangjunmiao accretionary complex and Yemaquan arc to post-collision in the East Junggar Orogen. Our results serve to better understand the gold mineralization and genesis of the Late Paleozoic orogenic system in the Kalamaili area, Xinjiang.