Based on the observation and analysis of cores and thin sections, and combined with cathodoluminescence, laser Raman, fluid inclusions, and in-situ LA-ICP-MS U-Pb dating, the genetic mechanism and petroleum geological significance of calcite veins in shales of the Cretaceous Qingshankou Formation in the Songliao Basin were investigated. Macroscopically, the calcite veins are bedding parallel, and show lenticular, S-shaped, cone-in-cone and pinnate structures. Microscopically, they can be divided into syntaxial blocky or columnar calcite veins and antitaxial fibrous calcite veins. The aqueous fluid inclusions in blocky calcite veins have a homogenization temperature of 132.5–145.1 °C, the in-situ U-Pb dating age of blocky calcite veins is (69.9±5.2) Ma, suggesting that the middle maturity period of source rocks and the conventional oil formation period in the Qingshankou Formation are the sedimentary period of Mingshui Formation in Late Cretaceous. The aqueous fluid inclusions in fibrous calcite veins with the homogenization temperature of 141.2–157.4 °C, yields the U-Pb age of (44.7±6.9) Ma, indicating that the middle-high maturity period of source rocks and the Gulong shale oil formation period in the Qingshankou Formation are the sedimentary period of Paleocene Yi'an Formaiton. The syntaxial blocky or columnar calcite veins were formed sensitively to the diagenetic evolution and hydrocarbon generation, mainly in three stages (fracture opening, vein-forming fluid filling, and vein growth). Tectonic extrusion activities and fluid overpressure are induction factors for the formation of fractures, and vein-forming fluid flows mainly as diffusion in a short distance. These veins generally follow a competitive growth mode. The antitaxial fibrous calcite veins were formed under the driving of the force of crystallization in a non-competitive growth environment. It is considered that the calcite veins in organic-rich shale of the Qingshankou Formation in the study area has important implications for local tectonic activities, fluid overpressure, hydrocarbon generation and expulsion, and diagenesis-hydrocarbon accumulation dating of the Songliao Basin.