In E. coli, FabA, a bifunctional enzyme, is the key enzyme of the classic anaerobic pathway of unsaturated fatty acid synthesis and introduces the cis double bond into a 10-carbon intermediate. This intermediate is then elongated by FabB (one of long chain 3-ketoacyl-ACP synthases) to form the unsaturated fatty acids found in the membrane phospholipids. Sequence alignments indicated that Sinorhizobium meliloti SmFabA and SmFabB are 60.6% and 61.1% identical to E. coli FabA and FabB, respectively. Further analysis showed that the conservative active-site histidine residue in EcFabA and the Cys-His-His catalytic triads in EcFabB, are also found in SmFabA and SmFabB. The genetic complementary revealed that SmfabA is able to restore the growth and the fatty acid synthesis of the E. coli temperature sensitive mutant CY57 at nonpermissive temperature under addition low concentration triclosan to inhibit enoyl-acyl carrier protein reductases. Moreover, SmfabB is able to complement temperature sensitive mutant CY242. In vitro assay identifies that SmfabA, like E. coli FabA, is able to introduce the cis double bond into a 10-carbon intermediate, and SmfabB, like FabB, is able to condense the acyl-ACPs with malonyl-ACP to long-chain acyl-ACPs. However, we also attempted to inactivate the fabA and fabB genes by allelic replacement but none of the fabA and fabB deletion mutant was obtained, and it seemed likely that fabA and fabB are essential genes in S. meliloti. These results demonstrated that SmFabA and SmFabB are key enzymes in unsaturated fatty acid synthesis in S. meliloti.