[Objective] Strain CICC 10774 causing vinegar spoilage was identified by polyphasic taxonomy and its growth and metabolism properties were studied. [Methods] Strain CICC 10774 was identified by 16S rRNA gene sequences phylogenetic analysis combined with morphology and physiological and biochemical characteristics. The optimal temperature and pH value for growth were measured using spectrophotometric method and the metabolic products were analyzed by HPLC. [Results] Polyphasic taxonomy analyses revealed that strain CICC 10774 was identified as Lactobacillus acetotolerans. The strain was Gram-positive, facultative aerobic, positive for gelatinase activities and could use mannose, N-acetylglucosamine, arbutin, esculin, salicin, cellobiose, trehalose and gentiobiose as sole carbon sources for growth. The optimal growth was observed at 37 °C and pH 5.0, and major metabolic products were acetic acid and lactic acid, indicating that strain CICC 10774 was a typical acidophilic bacterium. [Conclusion] The growth and metabolism properties of Lactobacillus acetotolerans CICC 10774 causing vinegar spoilage were studied to provide a theoretical basis for the vinegar production enterprises to control microbial pollution.