In this study, the IiEMF gene was first cloned based on the Isatis indigotica transcriptomic analysis. (1) Sequence analysis showed that the IiEMF gene contains an open reading frame of 1 896 bp encoding 631 amino acids. The phylogenetic analysis suggests that the IiEMF protein was relatively close to the EMF protein of Brassica oleracea. (2) Realtime quantity PCR (QPCR) resulted that IiEMF existed in all organs in I. indigotica. The highest expression level was observed in leaves and the lowest was in fruits. IiEMF expression level in the leaves increased first and then decreased and the highest peak emerged at first flower appearing stage. The IiEMF expression level in flower/fruit stage was significantly lower than that in flower bud stage. (3) The overexpression vector pCAMBIA1300EMF was constructed and then was introduced into A. thaliana by Agrobacterium. There are 7 strains of A. thaliana plants of transgenic EMF gene detected by PCR. (4) The results of phenotype observation indicated that the flowering time of the two transgenic EMF arabidopsis strains was significantly earlier than that of the wild type (6~10 d) under longday and shortday conditions. The rosette leave number of transgenic IiEMF gene lines was at least ten more than that of the wild type, and the leaves were also larger and thicker than that of wild type. (5) QPCR resulted that overexpression of IiEMF significantly inhibited the expression of AtAP1, AtCO and AtLFY and promoted the expression of AtFLC in the vegetative growth process of A. thaliana. The expression levels of AtAP1 and AtFLC in transgenic IiEMF gene lines were higher than that of wild type, and the expression levels of AtCO and AtLFY were significantly lower than that of wild type in reproductive growth period of A. thaliana. This study showed that overexpression of IiEMF promoted A. thaliana flowering and IiEMF may regulate A. thaliana flowering time by influencing multiple flowering pathways.