Objective To construct the overexpression vector and RNAi vector for porcine FSTL1 gene, and investigate the role and mechanism of FSTL1 gene on the differentiation of porcine preadipocytes.Method We constructed the overexpression vector and RNAi vectors of FSTL1 gene, transfected the vectors into porcine preadipocytes, and then induced differentiation of preadipocytes. Triglyceride content and the mRNA expression levels of adipocyte differentiation-associated genes were measured.Result The cDNA sequence of porcine FSTL1 gene was successfully cloned, the length of open reading frame was 924 bp, and the sequence has been submitted to GenBank with the login ID of KF021281. The overexpression vector pcDNA3.1-FSTL1 and the RNAi vectors pSilencer 4.1-491 and pSilencer 4.1-530 of the porcine FSTL1 gene were successfully constructed. Overexpressed FSTL1 gene promoted triglyceride content in porcine preadipocytes, whereas triglyceride content was reduced after FSTL1 gene was interfered. FSTL1 up-regulated the mRNA expression level of PPARγ2, AP2 genes by 73% and 113% respectively, and down-regulated LPL, Adiponectin and FASN genes by 44%, 79%, and 16% respectively.Conclusion The FSTL1 gene promotes the differentiation of porcine preadipocytes through up-regulating the mRNA expression of PPARγ2 and AP2 genes and down-regulating LPL, Adiponectin and FASN genes, and increases triglyceride content in porcine preadipocytes.