Abstract: Cold-seep bivalves obtain nutrients through symbiosis with chemosynthetic bacteria, and the biomarkers and stable carbon isotopic composition of their soft tissues can reveal their carbon sources, nutritional strategies, and symbiotic relationships with bacteria. Information derived from lipid biomarkers in modern cold-seep bivalves and their symbiotic bacteria is crucial for interpreting data from the rock record, as most biological methods are not applicable in such contexts. We applied lipid biomarkers and compound-specific stable carbon isotopic compositions to investigate the carbon assimilation pathways, nutritional status, and host-symbiont relationships of three cold-seep bivalves in the South China Sea, and focused on Acharax sp., Bathymodiolus aduloides, and Archivesica marissinica collected from the Haima cold seep, and analyzed the carbon isotopes and biomarkers in their soft tissues. Results show that the gills and feet of all the three cold-seep bivalve species contained significant amounts of saturated fatty acids, mono/polyunsaturated fatty acids (MUFAs/PUFAs), fatty alcohols, and sterols. Among them, the gill tissues exhibited much higher concentrations of C_16:1ω7, C_18:1ω7, and C_18:1ω9 compared to those in the feet, and the δ¹³C values were –35.2‰～ –30.8‰ for Acharax sp., –46.2‰～–45.7‰ for B. aduloides, and –42.9‰～–31.0‰ for A. marissinica. The soft tissues of these three cold-seep bivalves lack ω8 fatty acids and 4-methyl sterols, indicating that Acharax sp., B. aduloides, and A. marissinica rely on symbiosis with sulfur-oxidizing bacteria for nutrition. Additionally, the biomarkers in the feet of cold-seep bivalves and the animal-synthesized polyunsaturated fatty acid (C_20:2) exhibited δ¹³C values (–44.5‰ to –35.3‰) similar to those of symbiotic bacterial fatty acids, suggesting that the host’s nutrition was derived mainly from its symbiotic bacteria or that the majority of its carbon was acquired through this pathway. This study provided critical insights into the carbon utilization and symbiotic mechanisms of different cold-seep macrofauna.