AbstractQuorum‐sensing (QS) is an essential mechanism of microbial self‐regulation. In this work, the presence of QS in Acetobacter pasteurianus was demonstrated, and N‐acyl‐homoserine lactones N‐C4‐HSL and N‐C8‐HSL were identified as the signal molecules. The exogenous addition of N‐C4‐HSL, N‐C8‐HSL, N‐C10‐HSL, and N‐C12‐HSL result in the reduction in biomass, and lead to 11.56%, 11.91%, 7.94%, and 4.69% decrease in acetic acid production, respectively, when compared with the control. A hypothetical N‐acylhomoserine lactonase (AHL lactonase) 0046 was overexpressed in A. pasteurianus CGMCC 3089, and the recombinant strain exhibites a 17.38% and 12.75% increase in the biomass and the acetic acid production rate, respectively. Those results prove the negative regulation of QS on acetic acid fermentation, and overexpression of 0046 protein alleviates this negative effect.