A six-year (2010–2015) study of sugarcane (Saccharum officinarum) and soybean (Glycine max) intercropping combined with reduced nitrogen (N) fertilizer input was conducted to investigate sustainable agriculture via the functional soil microbial community in Southern China. The Illumina MiSeq platform and the microarray GeoChip 5.0 were applied in this study to detect the abundance, diversity and structure of bacterial and fungal communities, and to link specific functional genes with soil microbial-mediated N-cycling processes under experimental practice. The results revealed the α-diversity of bacterial communities to be relatively stable while the fungal α-diversity increased over the years. Intercropped sugarcane on rhizosphere soils under reduced and conventional N input harbor different bacterial and fungal community structures. Specifically, fungal α-diversity (e.g. Ascomycota and Basidiomycota) was found to be more sensitive to field practices while bacterial community (e.g. Bacteroidetes and Acidobacteria) changed moderately in abundance. Besides that, soil TN, AP, pH, and C/N ratio could explain the major variations in structure of soil microbial groups and in key functional genes. This study revealed the effect of N reduction and cropping patterns on functional traits based on both taxa and gene categories of microbial community, thereby improving understanding of structure-function relationships of microbial N-cycling processes in the different N input levels combined with cropping systems.