Aryl- O -demethylation is a common rate-limiting step in the catabolism of lignin-related compounds, including guaiacol. Here we used randomly barcoded transposon insertion sequencing (RB-TnSeq) in the bacterium Novosphingobium aromaticivorans to identify a Rieske-type guaiacol O -demethylase, GdmA. Similarity searches identified GdmA homologs in other bacteria, along with candidate reductase partners, denoted GdmB. GdmAB combinations were biochemically characterized for activity with several lignin-related substrates. Structural and sequence comparisons of vanillate- and guaiacol-specific O- demethylase active sites revealed conserved hallmarks of substrate specificity. GdmAB combinations were also evaluated in Pseudomonas putida KT2440, which does not natively utilize guaiacol. GdmAB from Cupriavidus necator N-1 demonstrated the highest rate of guaiacol turnover in vitro and in engineered P. putida strains and notably higher catalytic efficiency than a cytochrome P450 system (GcoAB) and the vanillate Rieske-type O -demethylase from P. putida (VanAB). The GdmAB O -demethylases described here expand the suite of options for microbial conversion of a model lignin-derived substrate. • A Rieske monooxygenase, GdmA, demethylates guaiacol in N. aromaticivorans DSM12444 • GdmA homologs and associated GdmB reductases display differences in specific activity • Structural comparison of O -demethylase active sites illuminates substrate specificity • Expression of GdmAB in engineered P. putida KT2440 enables growth on guaiacol Lignin is an abundant heterogenous component of plant cell walls, and its valorization is key to enable a profitable and sustainable global biofuels industry. A major challenge to convert lignin to chemicals is overcoming the inherent heterogeneity of deconstructed lignin polymers, and in recent years, microbial conversion has emerged as a means to produce single target compounds from mixtures of aromatic compounds. This work reports the discovery, characterization, and engineering of an enzyme system that can efficiently demethylate a lignin-related compound, 2-methoxy-phenol (commonly known as guaiacol). Aromatic O -demethylation is a common rate-limiting step in aromatic catabolic pathways and, thus, a prime target for discovery, engineering, and evolution of new enzymes. Going forward, biocatalysis development like that reported here may ultimately enable an integrated chemocatalytic and biocatalytic valorization approach for a major component of plant cell walls. Bacterial catabolism of lignin-related aromatic compounds is often limited by aryl- O -demethylation. This study identified a Rieske-type monooxygenase, GdmA, that catalyzes O- demethylation of guaiacol in Novosphingobium aromaticivorans. Detailed characterization of this enzyme, along with GdmA homologs and associated reductase partners (GdmB), identified differences in specific activity and substrate preference. Heterologous expression of GdmAB in Pseudomonas putida enabled growth on guaiacol. GdmAB O -demethylases expand the suite of options for microbial conversion of a model lignin-derived substrate.