Rotation practices have significant influences on the chemical, physical, and biological properties of soils, as well as on the occurrence of crop diseases caused by soil fungi. A long-term rotation experiment was set up in a red soil area of the Yunnan province with four rotation models: flue-cured tobacco-blank-maize (T-B-M), flue-cured tobacco-canola-maize (T-C-M), flue-cured tobacco-canola-rice (T-C-R), and flue-cured tobacco-vetch-rice (T-V-R). Chemical and enzymatic analysis and 454 pyrosequencing were used to study changes in nutrients, enzymes activities, and fungal colony structures in order to provide a scientific basis for the rational use of rotation of flue-cured tobacco. After 16 years of crop rotation, the soil pH changed from 5.6 to 6.4, which is still suitable for the cultivation of flue-cured tobacco, canola, rice, and maize. The yield, output value, and quality of flue-cured tobacco were increased significantly, and organic matter was increased by 45.11% under the condition of T-V-R treatment. Similar changes were also found in available nitrogen and phosphorus, soil microbial biomass carbon and nitrogen, and activities of sucrase, dehydrogenase, urease, phosphatase, and catalase. This suggests improvement of the soil ecological environment, promotion of microbial reproduction, and increases in microbial numbers and activities. The reads of fungal 18S rDNA sequences were 13097 in the soil with T-B-M, 11345 with T-C-M, 12939 with T-C-R, and 13763 with T-V-R, representing 530378395, and 581 fungal genera or species, respectively. All fungi belonged to Ascomycota, Basidiomycota, Zygomycota, Chytridiomycota, or unidentified groups, with the majority belonging to Ascomycota. The abundance of the top 15 predominant fungi accounted for 29.46%-62.86% of the total abundance, and less similarity was found in the tested soils, indicating variation in fungal colony structure in the soils with variable rotation models. T-V-R showed the highest Shannon diversity index but the lowest Simpson index of fungal communities, implying that the soils with T-V-R were favorable for the reproduction and growth of multiple fungi and increment of their groups. The co-presence of a variety of fungi in a soil could result in reciprocal inhibition and prevent their over-reproduction, thereby decreasing the possibility of the crops being infected by pathogenic fungi. With respect to crop yield, organic matter in soils, nutrients, and fungal community structure, T-V-R was superior to the other three rotation models, which could be encouraged in the tobacco-growing areas in Yunnan Province.