The phenomenon of sapropelic organic matter with high carbon isotope values, coupled with an abnormally high abundance of total organic carbon and hydrocarbon potential, has been identified in certain offshore lacustrine basins in China and rifting basins in the Atlantic. In this paper, we aim to investigate the mechanism behind the positive deviation of carbon isotope values within the organic matter of the Paleogene Wenchang Formation. Our research approach involves analyzing the organic carbon content, pollen, major and trace elements, as well as the carbon isotope and gas chromatograph- mass spectroscopy (GC- MS) of saturated hydrocarbon in both source rocks and crude oils.We also consider the characteristics of pediastrum in modern lakes to gain insights into the sources of organic matter, ancient environmental conditions, and the preservation and transformation of organic matter. The carbon isotope anomaly observed in the Wenchang Formation appears to be closely linked to various ancient environmental factors, including paleoclimate, paleoproductivity, and flourishing algae in the Zhu Ⅲ sub- basin. However, paleobathymetry may be a non- negligible significant factor on the scale of the third- order sequence.The Rb/K ratio, indicating the water depth, exhibits an increasing trend with depth, which coincides with the variations observed in carbon isotope values and TOC content. Additionally, modern lake investigations confirm the distribution of pediastrum across different water depths. Therefore, the bloom of “deep water type of pediastrum” is considered a key factor contributing to the high carbon isotope values observed in source rocks within the 2nd Member of the Wenchang Formation in the Zhu Ⅲ sub- basin.