The microorganisms surviving in ballast tank have to face very harsh conditions. After doing simulation experiment at land-based test facility for one month, high-throughput sequencing of 16S rRNA genes showed distinct differences between ballast water (test group) and natural water (control group), which indicated the mechanism that the bacterial communities might demonstrate temporal variations. A total of 413,806 high-quality sequences and 1,334 singleton operational taxonomic units were obtained. Proteobacteria and Bacteroidetes were the major phyla,but the richness of Proteobacteria in test group (mostly Gammaproteobacteria) was higher than that in control group. The concentrations of Acidobacteria and Chloroflexi gradually increased from day 15 in test group. At the family level, Flavobacteriaceae became the most abundant taxon on day 5, but gradually decreased thereafter in test group. And the abundance of Pseudoalteromonadaceae gradually increased and it became the dominant taxon from day 10 onwards. The UPGMA analysis showed a tighter clustering of the microbial composition of control group, indicating higher similarities in structure as compared to the test group samples which were more dispersed. PCoA and NMDS were also applied and the results showed that ballast tank environment impacted bacterial community structure.In this study, the richness and diversity peaked on the day 30 after ballasting, which was dissimilar to previous studies. An appreciable number of unclassified reads were noted at the genus level in test group samples during the later ballasting stage. More bacterial taxa were found in ballast water after long age post-ballasting than were present in natural seawater. This increase demonstrated that conditions inside ballast tanks could support the development of some taxa. It suggested that ballast tanks may act as incubators under special environments and further emphasized the risk of species invasion.