ABSTRACTGlucosamine (GlcN) is a widely utilized amino monosaccharide. It is traditionally synthesized from N‐acetylglucosamine (GlcNAc) via chemical processes that pose environmental threats. In pursuit of a greener alternative, our investigation explored biocatalysis with a Pantoea dispersa derived deacetylase (Pd‐nagA), showcasing its efficacy as a catalyst in GlcN production. As a result, this work provides a comprehensive characterization of Pd‐nagA, scrutinizes its enzymatic behavior, and delves into the deacetylation mechanism in detail. Heterologous expression methods were utilized for the production and isolation of Pd‐nagA, followed by a kinetic evaluation highlighting its enzymatic activity. The complex interactions between the enzyme and its substrate were investigated by integrating classical molecular dynamics, quantum mechanics/molecular mechanics simulations, funnel metadynamics, and on‐the‐fly probability enhanced sampling techniques, thereby elucidating the precise deacetylation pathway. Rigorous computational analysis results demonstrated that Pd‐nagA exhibited promising specificity and efficiency for GlcNAc with a high turnover rate. The catalytic residues central to the reaction were identified, and the underlying quantum reaction mechanism was detailed. Our findings suggest an approach to GlcN production using eco‐friendly biocatalysis, positioning Pd‐nagA at the forefront of industrial application not only because of its remarkable catalytic capabilities but also due to its potential for enzyme optimization.