Lactobacillus plantarum is a lactic acid bacterium that produces a racemic mixture of L- and D-lactate from sugar fermentation. The inter-conversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the L-/D-lactate ratio and maximally induced in the presence of L-lactate. We previously reported that the Lar activity depends on the expression of divergently orientated operons (larR(MN)QO and larABCDE). The larR gene codes for a novel transcriptional regulator of the Crp-Fnr family (PrfA group). Here, the role of LarR was further characterized in vivo and in vitro. We show that LarR is a positive regulator that is absolutely required for expression of Lar activity. Using gel retardation experiments, we demonstrate that LarR binds to a 16-bp palindromic sequence (Lar box motif) present in the larR-larA intergenic region. Mutations in the Lar box strongly affect LarR binding and completely abolish transcription from the larA promoter (PlarA). Two half-Lar boxes located between the Lar box and the -35 box of PlarA promote LarR multimerization on DNA and point mutations within one or both half-Lar boxes inhibit PlarA induction by L-lactate. Gel retardation and footprinting experiments indicate that L-lactate has a positive effect on binding and multimerization of LarR while D-lactate antagonizes the positive effect of L-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed.