Objective O-acetylhomoserine (OAH) is an important platform chemical to produce high-valuable chemicals. To improve the production ofO-acetylhomoserine from glycerol, the glycerol-oxidative pathway was investigated and the optimization of fermentation with crude glycerol was carried out. Results The glycerol-uptake system and glycerol-oxidative pathway were modified andO-acetyltransferase fromCorynebacterium glutamicumwas introduced into the engineered strain to produceO-acetylhomoserine. It was found that overexpression of glycerol 3-phosphate dehydrogenase improved the OAH production to 6.79 and 4.21 g/L from pure and crude glycerol, respectively. And the higher OAH production depending on higher level of transcription ofglpD. Two-step statistical approach was employed to optimize the fermentation conditions. The significant effects of glycerol, ammonium chloride and yeast extract were screened applying Plackett-Burman design and were optimized further by employing the Response Surface Methodology. Under optimized conditions, the OAH production was up to 9.42 and 7.01 g/L when pure and crude glycerol were used in shake flask cultivations, respectively. Conclusions The enzymatic step catalyzing the oxidation of glycerol through GlpD was the key step for OAH production, which served the foundation for realization of a consistent OAH production from crude glycerol in the future.