Abstract: In this study, three strains of Chlamydomonas, FACHB-2217 (Chlamydomonas reinhardtii), FACHB-479 (Chlamydomonas reinhardtii), and FACHB-889 (Chlamydomonas sp.)—were exposed to gradient concentrations of Hg²⁺ to investigate the molecular mechanisms underlying differential responses to mercury stress. The results showed marked differences in mercury tolerance among the three strains, with EC₅₀ values of 2.54, 1.09, and 0.85 mg/L, respectively. The tolerant strain FACHB-2217showed coordinated upregulation of ZIP, ABCB1, ABCC1, and ANT, facilitating Hg²⁺ compartmentalization and energy supply for low-cost homeostasis. The moderately tolerant FACHB-479 displayed impaired ZIP function and ANT blockade, leading to energy imbalance. The sensitive FACHB-889 exhibited extreme downregulation of DNA repair and carbon fixation genes, causing photosynthetic collapse and ROS imbalance.WGCNA identified a turquoise module correlated with mercury tolerance, with core genes including ABCB1, ABCC1, and multidrug and toxin extrusion protein (SLC47A). Mercury tolerance in Chlamydomonas is determined by multi-layered synergistic interactions among energy metabolism support, glutathione chelation, Hg²⁺ compartmentalization, and DNA/protein damage repair. These findings provide a theoretical basis for directed evolution or genetic engineering targeting key nodes such as ANT, equilibrative nucleoside transporter (SLC29A), ABCB1, and ABCC1.