Reversible phosphorylation of proteins is a critical mechanism involved in physiological function of organisms, including Clonorchis sinensis. In the present study, One cDNA clone encoding protein phosphatase 2A (CsPP2A) was isolated from a C. sinensis adult cDNA plasmid library. The open reading frame of the novel gene contains 924 bp and encoded a putative protein of 307 amino acids. A similarity analysis showed high homology with Schistosoma japonicum (76.3%) and Homo sapiens (84.4%), respectively. Recombinant CsPP2A (rCsPP2A) was expressed and purified from Escherichia coli BL21 using pET28a (+) as an expression vector. CsPP2A showed higher transcript level in adult worm but excysted metacercaria (P > 0.05), metacercaria (P < 0.05), and egg (P < 0.05) using real-time RT-PCR. Western blotting analysis showed that rCsPP2A could be identified by anti-rCsPP2A rat serum, C. sinensis-infected rat serum, and the serum from the rats immunized with excretory-secretory products of C. sinensis. Immunohistochemical assay showed that CsPP2A was deposited at the egg, the vitellarium of adult worm, and the excretory bladder of metacercaria. Collectively, the results of this study suggested that CsPP2A may be involved in the development of adult and metacercaria of C. sinensis.