Azithromycin (AZM), a widely used macrolide antibiotic, poses emerging risks to aquatic ecosystems, yet how it affects algal photosynthesis remains insufficiently resolved. Here, we investigated the physiological, structural, and transcriptomic responses of Chlamydomonas reinhardtii to AZM at environmentally relevant concentrations. Short-term exposure (72 h) revealed pronounced photosynthetic sensitivity, with significant reductions in Fv/Fm despite limited impacts on growth and cell integrity. Transcriptomic analysis showed concentration-dependent regulation of chlorophyll metabolism and photosynthetic pathways: low AZM enhanced expression of chlorophyll biosynthetic genes and induced compensatory upregulation of C4 cycle genes, while high AZM suppressed key enzymes (PBGD, PPOX) and photosystem genes, indicating impaired light harvesting and carbon fixation. Oxygen evolution assays identified the Mn₄ CaO₅ cluster as a primary target under low AZM and the plastoquinone pool under high AZM. Correspondingly, C. reinhardtii employed distinct photoprotective strategies: increased electron transport and photochemical efficiency at low AZM, and enhanced cyclic electron flow and non-photochemical quenching at high AZM. These findings reveal that AZM exerts concentration-dependent disruption of the photosynthetic apparatus, highlight PBGD, PPOX, and SGR as regulatory nodes, and underscore the value of sublethal photosynthetic endpoints in ecological risk assessment of antibiotics.