3-Ketoacy ACP reducatase (FabG), the key enzyme for bacteria growth, catalyzes the first reduction step in fatty acid synthesis. Pseudomonas putida has important application values in environmental pollution bioremediation and industrial polyhydroxy fatty acid (PHA) production. Bioinformatics analysis showed Pseudomonas putida genome encodes six FabG homologues with E. coli FabG, of which PpFabG5 shows the highest sequence similarity (76.5%) and other five PpFabGs also have high similarity (about 50%). Except PpFabG4, the other homologs have the conserved catalytic activity sites and N-terminal cofactor binding sites. So the paper used different methods, including the genetic complementary, catalytic activity analysis in vitro, gene deletion in vivo, and mutant characteristic analysis, to study the biological functions of the six homologs. The results showed that only PpfabG1, PpfabG3 and PpfabG5 could restore the growth of E.coli fabG temperature-sensitive mutant CL104 at 42℃, and the complementary strain of PpfabG1 grew weakly. While in vitro analysis, PpFabG1, PpFabG3 and PpFabG5 also showed the catalytic activities in the initial and extension reactions of fatty acid synthesis, although the activity of PpFabG1 was really weak. PpFabG6 only had catalytic activity in the initial reaction. PpfabG5 is an essential gene for growth, which can’t be deleted in the genome. While the deletion of the other PpfabG individually does not affect bacteria growth or the compositions of fatty acids comparing with wild type strain. However, the motility of PpfabG1 or PpfabG3 deletion mutant decreased, PpfabG3 or PpfabG6 deletion affected the biofilm formation, and PpfabG3 or PpfabG6 deletion mutant showed higher tolerance to H₂O₂. These results proved different FabG homologs contained different biological functions, especially when countered with stressful conditions.