The glycosyltransferase OleD variant as a catalyst for the glycosylation of four pairs of epimers of cardiotonic steroids (CTS) are assessed. The results of this study demonstrated that the OleD-catalyze glycosylation of CTS is significantly influenced by the configuration at C-3 and the A/B fusion mode. 3β-OH and A/B ring cis fusion are favoured by OleD (ASP). An epoxide ring at C-14 and C-15 further increases the bioconversion rate; while an acetyl group at C-16 and lactone ring type at C-17 did not influence the biotransformation. A high conversion rate corresponded to a low K_m value. A molecular docking simulation showed that filling of hydrophobic pocket II and interaction with residue Tyr115 may play an important role in the glycosylation reactions catalyzed by OleD glycosyltransferases. Furthermore, the glycosylation products showed a stronger inhibitory activity for Na⁺, K⁺-ATPase than the corresponding aglycones. This study provides the first stereoselective properties for OleD (ASP) catalyzed glycosylation.