Breakthroughs have been made recently in the exploration of hydrocarbon in the middle of Tarim Basin, NW China. The complexity of the Lower-Middle Ordovician carbonate reservoir remains enigmatic and hinders the source, developmental mechanism of the calcite fills, and paleo-environmental conditions of the Yingshan and Yijianfang Formations have been unclear. Here we present the related paleoenvironmental settings and diagenetic history, using petrographic, geochemical, and carbon (13C) and oxygen (18O) isotopic characteristics of calcite fracture, cave, and fissure fillings of a Lower-Middle Ordovician carbonate reservoir. The results indicate that the limestone caves dominantly consist of CaO, FeO, and MgO, and minor quantities of TiO2 and MnO. Among the trace elements, Ni reported the highest values (with an average of 1162.16 ppm) and other trace elements such as Ba, V, and Ga occur in minor values (with an average of 71.03, 19.18, and 12.92 ppm) respectively. The enrichment of light rare earth elements (LREEs) over the heavy rare earth elements (HREEs), with positive and negative Ce, and negative Eu anomalies indicate the contexts of oxic and anoxic conditions in seawater and sediments interface. The variation in δ18OPDB values ranges from −4.72 to −14.48‰ (with an average of −10.12‰), indicating a setting of paleo-karstification by atmospheric freshwater dissolution. While the variation in δ13CPDB indicates relatively significant values, ranging between 0.82 to −3.80‰ (with an average of −1.07‰), suggests that the paleo-karstification in this region has experienced episodes of supergene and burial diagenesis. The paleo-temperature average value of 59.1 °C, and the paleo-salinity average (Z-value) of 120.07, indicating a relatively warm paleoclimatic environment during the development of calcite-filling deposits. The results suggest several paleo-environmental conditions when paleokarst occurred: a marine deposition environment, an atmospheric freshwater karstification environment, a shallow burial paleo-karstification environment, and a high-temperature deep-burial environment in Yingshan and Yijianfang Formation in the Shunbei area. This study serves as an analog for similar carbonate systems worldwide, providing valuable insights for understanding paleo-karstification processes in different tectonic and climatic settings. Comparative studies between the Shunbei area, Tarim Basin, and other carbonate platforms contribute to our knowledge of karst evolution and its implications for reservoir development and groundwater resources.