Abstract: A 16-year field experiment has been carried out to investigate the influence of crop rotations on soil nutrients, microbial activities and bacterial community structures. The study includes 454 pyrosequencing analysis of bacterial 16S rDNA. The experiment investigated four crop rotation treatments: 1) flue-cured tobacco in summer, followed by winter fallowing and maize in summer (T-F-M); 2) flue-cured tobacco in summer, canola in winter and maize in summer (T-C-M); 3) flue-cured tobacco in summer, canola in winter and rice in summer (T-C-R); and 4) tobacco in summer, Vicia villosa in winter and rice in summer (T-V-R). After 16-years of crop rotation, soil pH had changed from 6.2 to 7.1. Compared with original soil levels, organic matter and available nutrients (such as nitrogen, phosphorus and potassium) either varied very little or significantly increased, suggesting the retention or growth of fertility under intensive rotations that return straw into soil and fallow during winter. There were higher microbial biomass carbon and nitrogen and enzyme activity (for example, sucrase, phosphatase, dehydrogenase, catalase and urease) in soils under the T-V-R treatment than in any of the other treatments. The readings of bacterial 16S rDNA sequences were 5395 for T-F-M soils, 5864 for T-C-M, 5528 for T-C-R and 6614 for T-V-R, representing 885, 973, 969 and 987 bacterial genera or species respectively, attributed to 31 phyla including Proteobacteria, Chloroflexi, Actinobacteria, Acidobacteria and Bacteroidetes. Proteobacteria were the largest group, accounting for 24.69%-32.49% of soil bacteria. 5 of the 31 bacterial phyla and 6 of the 15 predominant bacteria were found in all the treatments’ soils. It seems reasonable to suggest that crop rotation was a fundamental determinant of the abundance of soil bacterial components and community structures. Taking into account variations in organic matter, available nutrients, enzyme activities, microbial biomass, bacterial community structures and biodiversity, our experiment suggests that T-V-R could be the best rotation model to promote in the flue-tobacco cultivation areas of Yunnan province.