Abstract: This experiment was conducted to study the effects of different expression vectors and temperatures on the activity of recombinant Eimeria tenella Malonyl-CoA:ACP Transacylase (rEtMCAT). Using the ORF sequence encoding EtMCAT predicted by bioinformatic analysis as a template, 3 pairs of primers were designed in order to amplify the following fragments: the entire ORF, the ORF without N-terminal signal peptide, the ORF without N-terminal signal peptide and transit peptide，respectively. Each of the 3 cloned fragments was individually inserted into 3 plasmid vectors pMAL-c2x, pET-32a(+) and pProExHTa, respectively. All the recombinant vectors were transfected into E. coli Rosseta(DE3), and induced with IPTG under different temperature conditions (37, 30 and 16 ℃), respectively. The enzymatic activities of rEtMCAT were measured using the a-ketoglutarate dehydrogenase (KDH)coupled assay system. The results revealed that only the ORF encoding the mature peptide, that is, excised the N-terminal signal peptide and transit peptide could be expressed in 3 vector systems, and high content of soluble rEtMCAT was only expressed in recombinant plasmid pMAL-c2x-mcat or pET32a(+)-mcat, in which their expressed products had a similar enzymatic activity. The lower culture temperature (at 30 and 16 ℃) can enhance their expression. But there was no any soluble protein expressed in Rosseta(DE3)/pProExHTa-mcat, and the recombinant protein expressed in inclusion body was no enzymatic activity even after renaturation. The characterizations of expression vectors used in experiments, especially the type of tag protein, as well as the expression temperatures have significant effects on the production and enzymatic activity of rEtMCAT expressed in E. coli Rosseta(DE3). It is suggest that the rEtMCAT expressed in inclusion body can not be used for the enzymatic assay.