A ligand exchange of one of the acetonitrile ligands of the (acetonitrile)4Ru(II)-phenyloxazoline complex (Ru(II)-Pheox) by pyridine was demonstrated, and the location of the exchange reaction was examined by density functional theory (DFT) calculations to study the mechanism of its catalytic asymmetric reactions. The acetonitrile was smoothly exchanged with a pyridine to afford the corresponding (pyridine)(acetonitrile)3Ru(II)-Pheox complex with a trans orientation (C-Ru-N(pyridine)) in a quantitative yield, and the complex was analyzed by single-crystal X-ray analysis. DFT calculations indicated that the most eliminable acetonitrile is the trans group, which is consistent with the X-ray analysis. The direction of the ligand exchange is thus determined on the basis of the energy gap of the ligand elimination instead of the stability of the metal complex. These results suggested that a reactant in a Ru-Pheox-catalyzed reaction should approach trans to the C-Ru bond to generate chirality on the Ru center.