Aim To confirm the role of kinesin superfamily member 16B (KIF16B) in the process of low density lipoprotein cholesterol (LDLC) uptake of hepatocyte which is regulated by the inducible degradation of low density lipoprotein receptor (IDOL). Methods The intracellular fluorescence intensity was observed by the inverted fluorescence microscope. The intracellular lipid content was measured by oil red O staining, and the LDLC uptake was detected by DiI-LDL uptake experiment. The low density lipoprotein receptor (LDLR) abundances on the cell surface of hepatocytes were assayed by immune flow cytometry. The protein expression of IDOL, KIF16B and LDLR was detected by Western blot, and the interaction between LDLR and KIF16B protein was carried out by co-immunoprecipitation. Results Compared with white light view, the observed green fluorescence results showed that both HepG2 and LO2 cells were infected by the RNA-interference or overexpression IDOL(RNAi/OE-IDOL) lentivirus. Compared with the non-lentivirus infected control group, both the intracellular lipid and the ability of the LDLC uptake were significantly decreased in the OE-IDOL group(P＜0.05), and also decreased in the abundances of LDLR on the surface of hepatocytes (P＜0.01); and vice versa, the contrary results of these three experiments were observed in the RNAi-IDOL group (P＜0.01), which indicated that overexpression IDOL would reduce the LDLC uptake of hepatocytes. Compared with the RNAi/OE-IDOL control group, the expression of LDLR and KIF16B protein was increased in the RNAi-IDOL group (P＜0.01), and the interaction between KIF16B and LDLR was enhanced (P＜0.01). While in the overexpression IDOL of HepG2 and LO2 cells, the expression of LDLR and KIF16B protein was decreased (P＜0.05), meanwhile the interaction between LDLR and KIF16B was correspondingly weakened. Conclusion The interaction between KIF16B and LDLR possibly affects the process of that IDOL regulates LDLC uptake of hepatocytes.