The Southern Ocean is among the least explored marine environments on Earth, and still little is known about regional and vertical variability in the diversity of Antarctic marine prokaryotes. In this study, the bacterioplankton community in both epipelagic and mesopelagic waters was assessed at two adjacent stations by high-throughput sequencing and quantitative PCR. Water temperature was significantly higher in the superficial photic zone, while higher salinity and dissolved oxygen were recorded in the deeper water layers. The highest abundance of the bacterioplankton was found at a depth of 75 m, corresponding to the deep chlorophyll maximum layer. Both Alphaproteobacteria and Gammaproteobacteria were the most abundant taxa throughout the water column, while more sequences affiliated to Cyanobacteria and unclassified bacteria were identified from surface and the deepest waters, respectively. Temperature was the most significant environmental variable affecting the bacterial community structure. The bacterial community composition displayed significant differences at the epipelagic layers between two stations, whereas those in the mesopelagic waters were more similar to each other. Our results indicated that the epipelagic bacterioplankton might be dominated by short-term environmental variable conditions, whereas the mesopelagic communities appeared to be structured by longer water-mass residence time and relative stable environmental factors.