Calcineurin (CaN) is a Ca/calmodulin (CaM)-stimulated protein phosphatase. It is a heterodimeric enzyme consisting of a catalytic A subunit (CnA) and a Ca-binding regulatory B subunit (CnB). CaN's key role in vivo is well known, while the function of CnB keeps unclear except that it acts as a regulator of CaN. The present paper shows that CnB overexpression promotes proliferation of human embryonic kidney HEK293 cells by comparing with vector control cells in the complete or serum reduced medium. Furthermore, stable CnB transfectants showed dramatically improved growth in soft agar. And the migration ability of CnB overexpressors also was enhanced remarkably. But in the progress of transformation, the activity of CaN remained unchanged between CnB overexpressors and controls. Therefore, CnB, rather than CaN, is involved in the proliferation promotion of HEK293 cells. Subsequently, 11 proteins with different expression levels between CnB transfectants and controls were identified using two-dimensional gel electrophoresis and electrospray ionization time-of-fight mass spectrometry. Therein, the expression of heat shock protein 27 (HSP27) and protein DJ-1 increased along with CnB overexpression. The important role of CnB in cell neoplastic transformation was found and the possible mechanism was analyzed. © 2008 Japanese Cancer Association.