Ralstonia solanacearum, a soil-borne destructive plant pathogen, has an unusually wide host range, and causes a bacterial wilt that seriously affects the production of many economically important crops in the world. Thus, investigation of physiological metabolism in R. solanacearum will be helpful to develop new ways to control the bacterial wilt. Fatty acids are compulsory components of bacteria. However, the fatty acid biosynthestic mechanism is still unclear in R. solanacearum. In this paper, we identified acyl-CoA desaturase and cyclopropane fatty acid synthase of R. solanacearum GMI1000, and characterized their functions in biosynthesis of unsaturated fatty acid or cyclopropane fatty acid. First, R. solanacearum RSc2450 (desA) complemented Shewanella oneidensis desA fabA mutant growth on LB without supplemented oleic acid and caused E. coli fabA mutant to produce unsaturated fatty acid. Furthermore, deletion of desA caused R. solanacearum grows weak on BG plate and reduces the palmitoleic acid production. The results showed though R. solanacearum RSc2450 encodes an acyl-CoA desaturase and involves in unsaturated fatty acid biosynthesis, R. solanacearum might possess a novel unsaturated fatty acid biosynthetic pathway. Next, of the two putative cyclopropane fatty acid synthases encoded genes in R. solanacearum, only cfa1 (RSc0776) restored E. coli cfa mutant YYC1257 growth in low pH medium and to produce cis-9, 10-methylene palmitic acid. And deletion cfa1 mutant was sensitive to low pH and high osmotic pressure, and lost the ability to produce cis-9, 10-methylene palmitic acid. These indicated that cfa1 involves in cyclopropane fatty acid synthesis in R. solanacearum and plays roles in adaption to low pH and high osmotic pressure. Moreover, Cfa2 (RSp1446) does not have cyclopropane fatty acid synthase activity, and its function needs further study.