Microplastics (MPs) have caused increasing global concerns due to their detrimental effects on marine ecosystems. However, the role of photodegradation in altering toxicity of MPs to marine organisms is poorly understood. We therefore investigated the photolytic transformation of pristine polystyrene fragments (P-PS) by 60-day ultraviolet (UV) irradiation, and compared the toxicity of P-PS, photodegraded PS (PD-PS), and commercially available polystyrene microbeads (C-PS) to juvenile grouper (Epinephelus moara). Photodegradation reduced the size from ∼55.9 μm of P-PS to ∼38.6 μm of PD-PS, even produced nanoparticles (∼75 nm) with a yield of 7.03 ± 0.37% (w/w), and induced surface oxidation and formation of persistent free radicals (e.g., CO•, COO•). Also, endogenous pollutants (chemical additives and polymer fragments) were leached out. Thus, PD-PS had the highest growth inhibition and lipidosis-driven hepatic lesions of grouper, followed by P-PS and C-PS, which was mainly explained by increased hepatic bioaccumulation of MPs/NPs and released endogenous toxicants. Furthermore, oxidative stress-triggered mitochondrial depolarization, suppression of fatty acid oxidation and transport, and promotion of inflammation were identified as the key mechanisms for the enhanced hepatotoxicity after photodegradation. This work provides new insight into the potential hazard and harm of MPs in marine environments after photodegradation.