Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass, twenty hours after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence down-regulation of CETP, scavenger receptor class B type I (SR-BI), an HDL receptor, its expression was also reduced by about 50% in mRNA and protein levels, whereas, the apoA-I expression and secretion were increased by 30% and 92%, respectively. In a functional study, the selective uptake of ¹²⁵I-[¹⁴C]cholesteryl oleate/HDL₃ was decreased. Cholesterol efflux to apoA-I and HDL₃ was significantly increased by 88% and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about 2-fold increase of cholesterol esterification and increased ACAT1 mRNA levels. Taken together, these findings suggest that acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion and cellular cholesterol efflux to apoA-I, despite which, the return of HDL-CE to hepatocytes via a SR-BI pathway was inhibited in vitro. Thus, antisense inhibition of hepatic CETP expression manifests dual effects, namely increased formation of HDL and suppression of catabolism of HDL-CE probably via the SR-BI pathway.