Vitamin B₁₂ production by Propionibacterium freudenreichii sub sp. Shermanii was optimized using Taguchi orthogonal array (OA) design of experiment (DOE). An OA layout of L18 (2¹ x 3⁷) was constructed with eight most influencing factors on vitamin B₁₂ production, six factors like carbon, nitrogen, phosphate source, metal ion (Fe, Co) and inoculum size were selected based on ‘’one variable at a time approach’’ along with two other factors such as glucose (substrate) and DMBI (precursor). All the above factors except FeSO₄. 7H₂O (two level) were taken at three levels for proposed experimental design. Analysis was done by using Qualitek-4 software at bigger is better as quality character and obtained a predicted vitamin B₁₂ yield of 24.662 mg/L with a specific combination of factors. The optimal levels of factors (FeSO₄.7H₂O,12.5 mg/L; Inoculum size,10%; (NH₄)₂HPO₄, 0.1%; Glucose, 10%; DMBI, 0.025 g/L; Yeast extract, 1%, Sodium lactate, 1%; CoCl₂.6H₂O, 10 mg/L) obtained from designed methodology was further validated by confirmation experiment and the enhanced vitamin B₁₂ (16.2 mg/L to 23.1 mg/L) was obtained from its unoptimized condition. Taguchi approach of DOE methodology proved to be efficient in evaluating the interaction of each factor individually and in combination.