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Lipoic acid is an essential cofactor of several key enzymes involved in oxidative metabolism. The current model for protein lipoylation involves two pathways: one in which exogenous lipoate is transferred to apoproteins by lipoate ligase (LplA), and an endogenous one, that involves LipB, which transfers octanoate to target proteins. These octanoylated domains are converted into lipoylated derivatives by lipoyl synthase (LipA). We have previously constructed a Bacillus subtilis mutant deficient in lipA. In this work we report its use as a tool to study the interplay between branched-chain and unsaturated fatty acids (FA) metabolism in this organism. Interrupting lipoate-dependent metabolism strongly impairs the generation of branched chain FA and leads to the accumulation of copious amounts of straight-chain saturated FA into B. subtilis membranes. Although LipA depletion induces the expression of the D5-desaturase, the synthesis of unsaturated FA is insufficient to support growth in the absence of precursors for branched chain FA. However, deregulated overexpression of the D5-desaturase functionally replaces the deficiency in the lipoylated enzyme branched-chain 2-oxoacid dehydrogenase. This work reports the first characterization of a mutant deficient in lipoate synthesis in a Gram-positive organism and the role of this important cofactor in FA metabolism in B. subtilis.