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Formaldehyde is a known toxic compound, and functional formaldehyde detoxification is crucial for the survival of all living cells. Such detoxification systems are of particular importance for methylotrophic microorganisms that rely on formaldehyde as a central metabolite in their one-carbon metabolism. Understanding formaldehyde dissimilation pathways in non-methylotrophic industrial microorganisms is necessary for ongoing research aiming at engineering methylotrophy into their metabolism (synthetic methylotrophy). There is a variety of formaldehyde dissimilation pathways across microorganisms, often based on the activity of formaldehyde dehydrogenases. In this study, we investigated the role of the yycR gene of Bacillus subtilis putatively encoding a novel, uncharacterized zinc-type alcohol dehydrogenase-like protein. We showed that the B. subtilis ΔyycR mutant displayed a reduced formaldehyde tolerance level and confirmed the enzymatic activity of recombinantly produced and purified YycR as formaldehyde dehydrogenase in vitro. Biochemical analyses demonstrated that YycR activity is optimal at 40°C, with the highest measured activity at pH 9.5, formaldehyde is the preferred substrate, and the kinetic constants are Km of 0.19 ± 0.05 mM and Vmax of 2.24 ± 0.05 nmol min−1. Altogether, we showed that YycR is a novel formaldehyde dehydrogenase with a role in formaldehyde detoxification in B. subtilis, providing valuable insights for future research on synthetic methylotrophy in this organism. IMPORTANCE Formaldehyde is a key metabolite in methanol assimilation for many methylotrophic microorganisms, and at the same time, it is toxic to all living cells, which means its intracellular concentrations must be tightly controlled. An in-depth understanding of methanol detoxification systems in industrially relevant microorganisms is a prerequisite for the introduction of methanol utilization pathways into their metabolism (synthetic methylotrophy). Bacillus subtilis, an industrial workhorse conventionally used for the production of enzymes, is known to possess two formaldehyde detoxification pathways. Here, we identify a novel formaldehyde dehydrogenase in this bacterium as a path towards creating innovative prospect strategies for strain engineering towards synthetic methylotrophy [ABSTRACT FROM AUTHOR]