Despite the considerable efforts devoted to the toxicological assessment of nanoplastics, the effect of UV-irradiation induced aging, a realistic environmental process, on the toxicity of nanoplastics toward microalgae and its underlying mechanisms remain largely unknown. Herein, this study comparatively investigated the toxicities of polystyrene nanoplastics (nano-PS) and the UV-aged nano-PS on the eukaryotic alga Chlorella vulgaris, focusing on evaluating their inhibitory effects on carbon fixation. Exposure to environmentally relevant concentrations (0.1–10 mg/L) of nano-PS caused severe damage to chloroplast, inhibited the photosynthetic efficiency and electron transport, and suppressed the activities of carbon fixation related enzymes. Multi-omics results revealed that nano-PS interfered with energy supply by disrupting light reactions and TCA cycle and hindered the Calvin cycle, thereby inhibiting the photosynthetic carbon fixation of algae. The above alterations partially recovered after a recovery period. The aged nano-PS were less toxic than the pristine ones as evidenced by the mitigated inhibitory effect on algal growth and carbon fixation. The aging process introduced oxygen-containing functional groups on the surface of nano-PS, increased the hydrophilicity of nano-PS, limited their attachment on algal cells, and thus reduced the toxicity. The findings of this work highlight the potential threat of nanoplastics to the global carbon cycle.