Unsaturated fatty acid biosynthesis is a vital facet of Escherichia coli physiology and requires the expression of two genes, fabA and fabB, in the type II fatty acid synthase system. This study links the FabR (YijC) transcription factor to the regulation of unsaturated fatty acid content through the regulation of fabB gene expression. The yijC (fabR) gene was deleted by replacement with a selectable cassette, and the resulting strains (fabR::kan) possessed significantly elevated levels of unsaturated fatty acids, particularly cis-vaccenate, in their membrane phospholipids. The altered fatty acid composition was observed in the fabR::kan fabF1 double mutant pinpointing fabB as the condensing enzyme responsible for the increased cis-vaccenate production. The fabR::kan strains had 4- to 8-fold higher levels of fabB and a 2- to 3-fold increase in fabA transcripts as judged by Northern blotting, Affymetrix array analysis, and real-time PCR. FabR did not regulate the enzymes of fatty acid beta-oxidation. The elevated level of fabB mRNA was reflected by higher condensing enzyme activity in fabR::kan fabF1 double mutants. Thus, FabR functions as a repressor that potently controls the expression of the fabB gene, which in turn, modulates the physical properties of the membrane by altering the level of unsaturated fatty acid production.