3-ketoacyl ACP synthase Ⅲ (FabH) catalyzes the initial reaction in bacterial fatty acid synthesis. Gram-positive bacterial FabHs are able to utilize branched chain acyl-CoA as a primer, and are essential for the synthesis of branched chain fatty acids (BCFAs). However, some Gram-negative bacteria also synthesize BCFAs through a mechanism that is poorly understood. To compare the features of different bacterial FabHs, we selected four homologous Gram-positive bacterial fabH genes (Bacillus subtilis BsfabH1 and BsfabH2, Staphylococcus aureus SafabH and Streptomyces coelicolor ScofabH), and three homologous Gram-negative bacterial fabH genes (Escherichia coli EcfabH, Ralstonia solanacearum RsfabH, and Xanthomonas oryzae pv. oryzae XoofabH) for further study. Expression of each of the seven fabH genes restored the growth of the R. solanacearum fabH mutant RsmH in the absence of octanoic acid, indicating that all encoded FabHs have 3-ketoacyl ACP synthase Ⅲ activity. Furthermore, fatty acid profiling showed that complementation with the four Gram-positive bacterial fabH genes or the Gram-negative XoofabH rendered the R. solanacearum fabH mutant able to produce a large quantity of BCFAs, while the RsmH mutant harbouring EcfabH or RsfabH could only synthesize straight chain fatty acids, suggesting XooFabH performs a role different from EcFabH or RsFabH in BCFA sysnthesis. In vitro analysis showed that, similar to the four Gram-postive FabHs, XooFabH has a preferance for using branched chain acyl-CoAs as primers. XooFabH also displayed a strong activity towards short or medium chain acyl-CoAs (~C4-C10), unlike other Gram-positive FabHs. These results confirmed that FabH from different bacteria have different biological characteristics, and display a preferance for branched chain acyl-CoAs, which are essential for BCFAs synthesis.