Soil deterioration, yield decline and soil microbial activity reduction caused by banana (Musa × paradisiaca L.) monoculture is threatening the sustainability of banana production in China. Therefore, it is necessary to study the benefits of rotation on soil quality. This study aimed to assess the effects of rotation and tillage on soil properties in a banana plantation for 25 yr. Treatments consisted of three rotation methods (banana-pineapple, BA; banana-cowpea, BP; banana-rice, BR) and banana monoculture (CK) combined with two tillage intensities (no-tillage, NT; conventional tillage, CT). Soil samples were taken at depth of 0-40 cm in 2019-2020. In comparison with CK, BA and BR increased soil moisture, pH, total organic C and available P, but decreased soil bulk density. Microbial biomass C and N at booting stage were 46.1% and 39.2% higher in BA and BR than those in CK. Urease, dehydrogenase and β-glucosidase obtained a mean of 34.1% increase in BP and 23.8% increase in BR compared with BA. Higher total N, NO3-N, available K and macroaggregate were showed in NT compared with CT, whereas porosity was 24.8% lower in NT than in CT. CO 2, N2O and CH4 emissions were in average around between one third and two fifth lower in no-tillage compared with conventional tillage. In general, rotations combined with no-tillage led to a positive effect on soil quality, as evidenced by increase of soil moisture, total N, microbial biomass C and urease and accompanying increase in banana yield. In order to sustain higher productivity, application of rotation and no-tillage is of considerable importance.