The large Jiulong calcite deposit, located at the northwest part of Central Yunnan Paleozoic Basin in southwest margin of the Yangtze platform, is the only giant crystal calcite deposit discovered so far in central Yunnan Province, SW China. The ore genesis is still unclear. In this paper, based on geological investigation and petrographic observation, the giant crystal calcite ore zone, dolomitic rock zone, and mottled calcilization rock zone are selected to carry out mineral chemistry, C-O isotopic composition and fluid inclusion analysis of different types of calcites and ore-hosting rocks, and calcite U-Pb dating. The results show that the proved orebodies are mainlyhosted along the beddings of the hosting limestones in the stratiform shape, and others exist in the bedding horizons and steeply dipping fault fracture zones in the form of large vein types. Both limbs of the NE-SW-extending Wenlin syncline and the transitional segments of the secondary "dome-and-basin" structure are favorable ore-depositional areas. And the middle and upper successions of the Lower Permian Maokou Formation (P₁m) limestone are the main ore-hosting rocks. The Jiulong calcite deposit is of low-temperature epigenetic origin deposit with prominent features of hydrothermal fluid replacement and filling and ore-structure controlling. The ore-forming fluid is featured by the Cl-Na·Ca type basin brine, and ore-formation materials and fluids are mainly originated from the cyclic interaction between the ore-hosting sediments and underlying basement sequences. There may be the addition of deep magmatic hydrothermal fluid. The small temperature variation, slow nucleation rate, and long-term crystallization process are the key mechanism of the ore-formation of giant crystal calcites. The metallogenic age of this deposit is 36.0±4.0 Ma, that is the Eocene-Oligocene times. Therefore, the ore-formation of the Jiulong calcite deposit is the evolutionary product of the regional tectonic-controlling basin fluid during the Himalayan age, representing the remote respondence to the Cenozoic Indo-Eurasian collisional orogeny in the Central Yunnan Paleozoic Basin.