Lignin is an abundant and complex aromatic polymer found in plants comprised predominately of p-coumaryl (H type), coniferyl (G type), and sinapyl (S type) alcohol subunits. The biological valorisation of lignin offers a green alternative to the production of commercially useful products which have been traditionally synthesised from unsustainable sources. Developments in lignin depolymerisation have provided the opportunity for lignin-derived aromatic compounds (LDACs) to be directed towards chemically useful products. However, a roadblock towards efficient lignin valorisation is the availability of enzymes which catalyse the rate limiting O-demethylation of LDACs. Several cytochrome P450 O-demethylases have been characterised that act on G and H type LDACs [1]. However, no native S type O-demethylase has previously been identified. Thus, the bioconversion of LDACs derived from S lignin – which is abundant in hardwood and grasses - is limited. In this work, we have characterised a novel bacterial cytochrome P450 enzyme from Amycolatopsis thermoflava capable of catalysing the O-demethylation of syringol – a major LDAC of S type lignin. Homology modelling and in silico docking were used to determine the binding specificity of the cytochrome P450 and in vitro UV/vis spectroscopy and hydrogen peroxide turnovers were used to verify binding and catalysis. The discovery and characterisation of S type lignin O-demethylases will enable the development of engineered microbes for the efficient valorisation of heterogeneous lignin to commercially useful products.