Microbial acetate metabolism is an important part of marine carbon cycling. We present a comprehensive study to constrain microbial acetate metabolism and its regulation in surface seawater of the northwest Pacific Ocean. We found that acetate oxidation (rate constant k: 0.016–0.506 day−1) accounted for 77.6%–99.4% of the total microbial acetate uptake, suggesting that acetate was predominantly used as a microbial energy source. Acetate also served as a significant biomass carbon source, as reflected by the elevated contribution of acetate assimilation to bacterial carbon production. Acetate turnover was largely influenced by water mass mixing and nutrient conditions. Atmospheric deposition was a source of acetate in surface water and this process can also impact the microbial acetate uptake. Microbial utilization of acetate could account for up to 25.9% of the bacterial carbon demand, suggesting the significant role of acetate metabolism in microbial carbon cycling in the open ocean.