[Background] Streptomyces strains are widely distributed in soil environment. With a complex morphological differentiation and a large diversity of secondary metabolic networks, Streptomyces can produce many bioactive secondary metabolites. [Objective] Fatty acid biosynthesis and secondary metabolism are closely related in the model strain Streptomyces coelicolor, but the fatty acid synthetic mechanism is still unclear, and its long-chain 3-ketoacyl ACP synthase has not been reported. [Methods] Through sequence alignment with Escherichia coli FabF (EcFabF), SCO2390 (ScoFabF1), SCO1266 (ScoFabF2), SCO0548 (ScoFabF3) and SCO5886 (ScoRedR) were found in the genome of Streptomyces coelicolor A3(2), which showed high similarity with EcFabF, and contained the conserved Cys-His-His sites, indicating that they may have the 3-ketoacyl-ACP synthase activity. The four genes were amplified by PCR, and ligated into the expression vector pBAD24M, and transferred into E. coli fabB(ts) and E. coli fabB(ts)fabF mutants. The growth of transformants was analyzed. The four genes were also ligated into pET-28b, and expressed in E. coli BL21(DE3). The four EcFabF homologues with hexahistidine-tag were purified by Ni-NTA, and the activities were analyzed in vitro. The fatty acid profiles of E. coli fabF mutants completed with the four genes were also analyzed by GC-MS. [Results] Only ScofabF1 conferred the E. coli fabB(ts)fabF mutant to grow with oleatic acid supplemented at 42 °C, and all failed to complete E. coli fabB(ts) at 42 °C. In vitro enzymatic analysis also demonstrated that only ScoFabF1 has 3-ketoacyl-ACP synthase activity, while the other three proteins showed no similar activity. E. coli fabF mutant harboring ScofabF1 increased the amount of unsaturated fatty acid C18:1 significantly. [Conclusion] All of above suggested that Streptomyces coelicolor ScofabF1 encodes 3-ketoacyl-ACP synthase II, and plays an important role in fatty acid synthesis. However, no gene encoding 3-ketoacyl-ACP synthase I was found in the genome, indicating that Streptomyces coelicolor may have other mechanism to synthesize small amount of unsaturated fatty acids. Achievement in this study will contribute to further research about the mechanism of fatty acid synthesis in Streptomyces coelicolor.