Enantioselective partitioning of racemic omeprazole enantiomers was studied using a biphasic recognition chiral extraction system. Hydrophilic hydroxypropyl-ڂ-cyclodextrin in aqueous phase and hydrophobic D-tartaric acid hexyl ester in organic phase as chiral selectors which preferentially recognize (R)-omeprazole enantiomer and (S)-omeprazole enantiomer, respectively. Different experimental variable parameters could affect the chiral extraction efficiency. The largest distribution coefficientskS,kRand separation factorځwere obtained at concentrations o f 0.1 mol/L HP-ڂ-CD and 0.2 mol/L D-tartaric acid hexyl ester, which were 47.38, 58.65 and 1.24, respectively.kRis always larger thankSwhen using different kinds of tartaric acid derivatives as chiral selectors in organic phase. The present study also reveal the mechanism of biphasic recognition chiral extraction forR,S-omeprazole enantiomers.