Abstract: Banana wilt caused by Fusarium oxysporum f. sp. cubense is one of the most widespread and destructive plant diseases in the world. To date, the efficient prevention and control of F. oxysporum in banana have not been found. Recently, banana-sugarcane rotation plantation (recorded in the Chinese ancient book Guangdong New Sight) has been noted to have the potential to control the disease. Furthermore, preliminary filed studies showed that the incidence of banana wilt disease reduced from 49.15% to 1.79% after two years of planting sugarcane in continuous cropped banana fields. In this study, the control effect and mechanism of banana-sugarcane rotation was investigated. Banana wilt disease incidence under various years (one, two and three years) of banana replanting after two years of sugarcane rotation were investigated. The number of soil culturable micro-organisms was measured and soil microbial community structure analyzed by high-throughput DNA sequencing technique. The results showed that banana wilt disease incidence was only 1.79% in first replanted banana field after two years of sugarcane rotation. Then it was respectively 21.93%, 25.80% and 28.81% in the second, third and fourth years of banana replantation; less than that under continuous cropping of banana fields (49.15%). The number of F. oxysporum and Actinomycetes significantly increased with increasing banana replantation years. Sequence analysis showed that Acidobacteria (32.86%), Proteobacteria (28.85%) and Chloroflexi (12.33%) constituted the dominant phyla in the banana-sugarcane rotation system. The relative abundance of bacteria of Acidobacteriales apparently increased with increasing banana replantation years and had a significant positive correlation with the incidence of banana wilt. Meanwhile, the bacteria number of Pseudomonadales, Planctomycetales, Sphaerobacterales, Lactobacillales and Solirubrobacterales apparently decreased and were significantly negatively correlated with the incidence of banana fusarium wilt. In conclusion, the control effect was still good in terms of replanting banana in less than four years after two years of sugarcane rotation. The best cultivation system was shorter rotation time, longer duration disease inhibition and higher economic efficiency, compared with other banana rotation patterns. The results showed that the inhibition of Acidobacteriales and the promotion of Pseudomonadales, Planctomycetales and Solirubrobacterales were the most important mechanisms to reduce the incidence of banana fusarium wilt under banana-sugarcane rotation systems.