Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) fromLactobacillus acidophilus. Our results demonstrated thatLaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhanceLaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration ofLaFae, crystal structures of unliganded and ethyl ferulate-boundLaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase inLaFae activity againstp-nitrophenyl butyrate. Our results highlight the considerable application potential ofLaFae to produce ferulic acid from plant biomass and agricultural by-products.