In this study, the growth and metabolic characteristics of Acetobacter pasteurianus and Lactobacillus plantarum used in the co‐fermentation of apple vinegar were investigated. The results revealed that L. plantarum had little effect on the growth of A. pasteurianus during the co‐fermentation process. The carbon sources used by L. plantarum and A. pasteurianus for growth were glucose and ethanol, respectively. Free amino acids, organic acids, and acetoin were analyzed using an amino acid analyzer, high‐performance liquid chromatography, and gas chromatography, respectively. The results indicated that L. plantarum contributed to the accumulation of free amino acids during co‐fermentation. Lactic acid, a key flavor substance, was accumulated mainly by L. plantarum during co‐fermentation and was partly transformed into acetoin by A. pasteurianus. The acetoin content was 0.77 mg/ml, which is about twice that observed during pure fermentation with A. pasteurianus. Therefore, L. plantarum can improve the quality of apple vinegar during co‐fermentation. Novelty impact statement L. plantarum had little effect on A. pasteurianus growth during co‐fermentation of apple vinegar. The free amino acid and organic acid content in apple vinegar increased considerably because of the growth and metabolism of L. plantarum. The metabolites from L. plantarum could also be used by A. pasteurianus to improve the production of flavor substances during co‐fermentation.