E. coli is well studied in fatty acid synthesisⅡ. FabA, a bifunctional enzyme, is the key enzyme of the classic anaerobic pathway of unsaturated fatty acid synthesis. Sequence alignments indicated that Lactococcus lactis lacks homologues of E. coli FabA, however it owns two homologues of E. coli FabZ, LlfabZ1 and LlfabZ2. LlFabZ1 and LlFabZ1 are 41% and 45.1% identical to E. coli FabZ, respectively. Further analysis showed that the conservative active-site in E. coli FabZ is also found in LlFabZ1 and LlFabZ1. Although the genetic complementary revealed that LlfabZ1 and LlfabZ2 were not able to restore the growth and the fatty acid synthesis of the E. coli temperature sensitive mutant CY57 at nonpermissive temperature, cell-free extract data showed LlFabZ1 was able to catalyze the isomerization of the trans-2-double bond to the cis-3 species. Moreover, LlfabZ2 was able to complement the EcfabZ knock out mutant HW7. In vitro assay identified that LlFabZ1 was able to introduce the cis double bond into a 10-carbon intermediate, and LlFabZ2 was able to dehydrated 3-hydroxyacyl-ACP to trans-2-decenoy-ACP. However, we also attempted to inactivate the fabZ1 and fabZ2 genes by allelic replacement but none of the fabZ1 and fabZ2 deletion mutant was obtained, and it seemed likely that fabZ1 and fabZ2 are essential genes in L. lactis. These results demonstrated that LlFabZ1 and LlFabZ2 are key enzymes in fatty acid synthesis in L. lactis.