Aim To evaluate the potential inhibition of lipoprotein lipase (LPL), the rate limiting enzyme in hydrolyzing plasma triglyceride, in cultured cells by RNA interference technique, which would be further explored as a new way for intervention of atherosclerosis. Methods Two target sequences were designed according to human LPL sequence information and cloned into a plasmid expressing desirable small hairpin RNA (shRNA) in mammalian cells. This plasmid was co-transfected with LPL expressing plasmid into COS-7 cells. Real time polymerase chain reaction (PCR), Western blot and enzyme activity assay were carried out to determine the inhibition of LPL gene expression. Results Compared with the control cells, more than 70% inhibition of LPL gene expression in cells co-transfected with shRNA was observed at the levels of mRNA, protein and enzymatic activity. Conclusion Expression of LPL gene can be efficiently inhibited in vitro by RNAi technique. These results are informative for further study in vivo.