To obtain a mutant with higher vinegar production, a wild-type industrial strain Acetobacter pasteurianus CICIM B7003 was pretreated in 50 g L−1 acetic acid for 1 h and cultured for 12 h without adding any acetic acid; then, the enriched cells were mutated by UV under acidic stress (60 g L−1 acetic acid). Using this method, A. pasteurianus CICIM B7003-02, a mutant exhibiting best performance, was obtained. Notably, after repeated experiments, it was confirmed that B7003-02 accumulated 103.81 ± 1.17 g L−1 acetic acid within 160-h batch cultivation in Erlenmeyer flasks, which was 49.2% higher than that of the wild type. Repeated batch fermentations with A. pasteurianus B7003-02 were carried out for several runs in a Frings 8-L acetator, and high-acidity vinegars (90 ± 0.39 g L−1) were produced. This work reveals the potential value for improvement in industrial vinegar production.