Methylobacterium sp. MB200 capable of producing glyoxylate from methanol was obtained by enrichment culture using a medium containing methanol as the sole carbon source. A hpr gene that encodes a hydroxypyruvate reductase (HPR) was cloned from this strain and was ligated into the vector pLAFR3 to obtain the recombinant plasmid pLAFRh, which was transferred into M. sp. MB200 to generate an recombinant strain MB201. Homologous expression of hpr under the control of the lacZ promoter led to the enhanced glyoxylate accumulation in cultures of Methylobacterium sp MB201. The yield of glyoxylate reached 14.38 mg/mL, representing nearly a twofold increase when compared with the wild-type strain.