In this study, the ability of Tinospora crispa aqueous extract (TCAE) to regulate cholesterol metabolism in human hepatoma cancer cell line (Hep G2) was determined. Cytotoxic study was performed by exposing hepatoma cell (Hep G2) towards TCAE with concentration ranging from 0.002 to 20 mg/ml for 24 h at 37°C and with 5% CO 2 atmosphere. Result revealed that TCAE was not toxic to the cell. The ability of TCAE to reduce cholesterol in cell culture experiment was carried out by pre-treating Hep G2 with selected concentrations of TCAE (10, 5, 2.5, 1.25 and 0.625 mg/ml) in 6-well plate before the cell was exposed to low density lipoprotein (LDL). The concentration of apolipoprotein A1 (Apo A1), lecithin-cholesterol acyltransferase (LCAT), low density lipoprotein receptor (LDLR), scavenger receptor B1 (SRB1) and hepatic Lipase (HL) which involve in reverse cholesterol transport (RCT) pathway were determined from the 6-well plate medium. The direct pathway of cholesterol synthesis was performed according to the instruction provided in HMG-CoA Reductase Assay Kit manuals. The results showed that TCAE significantly increase (p<0.05) the concentration of Apo A1, LCAT, LDLR, SRB-1 and HL. The efficacy of these activities is appreciably good when compared with standard drug simvastatin. However, TCAE showed moderate effect in controlling mevalonate pathway. These findings suggested that TCAE has the potential to reduce cholesterol metabolism in Hep G2 cancer cell lines and the pathway of TCAE action possibly more on RCT.