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Zinc Oxide Nanoparticles (ZnO NPs) have attracted increasing concerns because of their widespread use and toxic potential. In this study, Zn accumulations in different tissues (gills, liver, muscle, and gut) of goldfish (Carassius auratus) after exposure to ZnO NPs were studied in comparison with bulk ZnO and Zn(2+). And the technique of subcellular partitioning was firstly used on the liver of goldfish to study the hepatic accumulation of ZnO NPs. The results showed that at sublethal Zn concentration (2 mg/L), bioaccumulation in goldfish was tissue-specific and dependent on the exposure materials. Compared with Zn(2+), the particles of bulk ZnO and the ZnO NPs appeared to aggregate in the environmentally contacted tissues (gills and gut), rather than transport to the internal tissues (liver and muscle). The subcellular distributions of liver differed for the three exposure treatments. After ZnO NPs exposure, Zn percentage in metal-rich granule (MRG) increased significantly, and after Zn(2+) exposure, it increased significantly in the organelles. Metallothionein-like proteins (MTLP) were the main target for Zn(2+), while MRG played dominant role for ZnO NPs. The different results of subcellular distributions revealed that metal detoxification mechanisms of liver for ZnO NPs, bulk ZnO, and Zn(2+) were different. Overall, subcellular partitioning provided an interesting start to better understanding of the toxicity of nano- and conventional materials.