Copper (Cu) is one of the most important essential metals for crustaceans, buttoxic in excess. Metallothioneins (MT) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis, detoxification, and cytoprotection. In the present study, Sinopotamon henanense were exposed to 0 (controls), 2.86, and 14.3 mg L−1 waterborne Cu, Cu accumulation, zinc (Zn) levels and MT induction in gills and hepatopancreas were determined with Cd/Hemoglobin saturation assay and atomic absorption spectrophotometry method. Results showed that Cu accumulation and MT levels were both tissue-specific and revealed some time-dependent and dose-dependent, respectively. The highest Cu accumulations of 82.10 ± 16.38 μg g−1 w wt were observed in the gill after 15 days of 14.3 mg L−1 Cu exposure, the peak MT induction of 136.16 ± 19.39 μg g−1 w wt were observed in the hepatopancreas after 3 day of 14.3 mg L−1 Cu exposure.In addition, the essential metal homeostasis of Zn was disturbed in some ways by subacute Cu exposure. The calculated ratios of actual Cu to theoretical maximum metal bound by MT indicating that the hepatopancreas had much greater Cu-binding potentials than the gills. Positive correlation were shown between MT induction and Cu accumulation both in hepatopancreas and gills, indicating that MT induction in S. henanense can be considered as a biomarker for subacute waterborne Cu pollution. Furthermore, the Cu induced MT (CuMT) from S. henanense was purified using acetone precipitation (50–80%), followed by gel filtration chromatography and anion exchange chromatography. SDS-PAGE and time-of-flight mass spectrometry analysis showed that S. henanense CuMT possess two isoforms and both mainly existed as monomer and dimmer forms. These present studies will be helpful to increase the database information of heavy metal-induced MT in terms of crustaceans. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 407–417, 2014.