Heterotrophic bacteria are the major consumers of organic carbon in the ocean. Accurate estimation of bacterial carbon metabolic rates is crucial for understanding the metabolic state in marine ecosystems. In order to further explore whether the bottle incubation can lead to bacterial growth and to quantify the overestimation of respiration rate obtained by the Winkler method, the bacterial dynamics in bottles during 24 h incubation were observed in this study during a total of 4 voyages in the Taiwan Strait (TWS) and the South China Sea (SCS) from 2018 to 2021. The results showed that the bacterial abundance (BA) and bacterial production (BP) increased 1.4- and 8.3-fold on average after incubation, respectively. The increase in BP was mainly attributed to the increase in cell-specific bacterial productivity. Comparing the estimated bacterial respiration (BR) from BP collected before incubation with BR measured by the Winkler method, we found that BR obtained by dark bottle incubation was overestimated by an average of 3.6-fold. The overestimation of BR in TWS was significantly higher than in SCS, and there was more overestimation in spring than in summer. Temperature and nutrient concentration were the key environmental factors influencing the overestimation of BR during incubation. Assessment results from the global mixed layer dataset showed that the overestimation of respiration by Winkler method may be a potential cause of the negative net community productivity (NCP) measured in oligotrophic ocean. Recognition of this result underscores the variability of respiration rates measured by bottle incubation and highlights the necessity to correct respiration data in order to figure out the truth of metabolic state controversy.