Glycolaldehyde (GA) was shown to be a precursor of vitamin B6 (B6), and to be formed from glycolate by glycolaldehyde dehydrogenase (GADH) in Escherichia coli. In this study, we show the glycolaldehyde-forming route in B6 biosynthesis in Bacillus subtilis. In the crude extract of B. subtilis, the oxidizing activity of GADH was detected. However, coexisting NADH/NADPH oxidase activity interfered with the determination of the reducing (GA-forming) activity of GADH. NADH/NADPH oxidase was purified and identified as the product of ahpF. In an ahpF disruptant, NADH/NADPH activity was almost eliminated, but the reducing activity of GADH was not detected. We also investigated another possible GA-forming enzyme, glyoxal reductase (GR). GR was partially purified and identified as the product of yvgN. yvgN disruptant did not require B6, and retained the ability to synthesize the same amount of B6 as the wild-type strain. From these results, we concluded that neither GADH nor GR is involved in B6 biosynthesis in B. subtilis.