7α-hydroxysteroid dehydrogenase (7α-HSDH) can catalyse the oxidation of C7 α-OH of the steroid nucleus in the bile acid metabolism. In the paper we determined the crystal structure of 7α-HSDH from Clostridium absonum (CA 7α-HSDH) complexed with taurochenodeoxycholic acid (TCDCA) and NADP(+) by X-ray diffraction, which, as a tetramer, possesses the typical α/β folding pattern. The four subunits of an asymmetric unit lie in the fact that there are the stable hydrophobic interactions between Q-axis-related subunits. Significantly, we captured an active state of the NADP(+), confirming that nicotinamide moiety of NADP(+) act as electron carrier in the dehydrogenation. On the basis of crystal structure analysis, site-directed mutagenesis and MD simulation, furthermore, we find that the guanidinium of Arg38 can form the stable cation-π interaction with the adenine ring of NADP(+), and the cation-π interaction and hydrogen bonds between Arg38 and NADP(+) have a significant anchor effect on the cofactor binding to CA 7α-HSDH.