Obesity and its related metabolic diseases become major health problems in the world. Adipose tissue plays an important role in the development of obesity. FSP27, a member of the CIDE family proteins, is expressed at high levels in white adipose tissue and differentiated 3T3L1 cells. The objective of current study is to establish a FSP27 knockdown preadipocyte cell line to investigate the in vivo function of mouse FSP27. The double strand siRNA of mouse FSP27 corresponding to nucleotides 270 to 291 was synthesized and inserted into pSilencer2.1. pSilencer-siFSP27 was co-transfected into 293T cells with the HA-mFSP27 expression vector to test its knock-down efficiency. The FSP27 siRNA was then transferred to a lentiviral vector. Lentivirus were generated and used to infect 3T3-L1 cells. It was shown here that lentivirus containing FSP27siRNA can effectively knockdown FSP27 expression in 3T3-L1 cells. Establishment of FSP27 knock-down cell line provides a useful tool for the study of in vivo function FSP27.