Aim To construct the soluble epoxide hydrolase(sEH) gene-specific recombinant vectors pSUPER retro neo,and selectively knockdown the expression of sEH in mice cardiomyocytes by RNA interference(RNAi),and to select the plasmids having the best silence effect to sEH. Methods Two pairs of siRNA that target at sEH gene were designed,and to construct the siRNA expression vectors specific to sEH gene(EH-1 and EH-2).There were plasmids carrying a nonspecific siRNA coding sequence(pCN) as the negative control group and the blank control group involved.Then the plasmids were transfected into primary cultured mice cardiomyocytes by FuGENE HD.The mRNA and protein expressions of sEH were analyzed by RT-PCR and Western Blotting respectively. Results The expressions of mRNA and protein of sEH in EH-2 group(0.202±0.017 and 0.212±0.029,P<0.01) were significantly decreased compared with that of blank control group,negative control group,and EH-1 group. Conclusion Recombinant expression vectors have been constructed,and RNA interference can selectively knockdown sEH expression in cultured cardiomyocytes.This lays a foundation for further research of RNAi on cardiomyocytes.