© 2016 Elsevier B.V. The addition of zinc (Zn) to the soil has significant gains in sugarcane productivity. The understanding of the consequences of zinc application requires insight into ecological aspects of changes in soil microbial communities. Here, we evaluated the effects of zinc application on richness, diversity, evenness and structure of microbial functional genes in sugarcane-cultivated soil, and response thresholds of abundance for groups of microbial functional genes on different zinc concentrations added to the soil (0, 5, 10 and 20 kg Zn ha−1) in two sugarcane fields in the Northeastern São Paulo state, Brazil. Using a high-density functional gene array termed GeoChip 5.0, which contains 101,796 distinct probes belonging to gene categories involved in zinc transport, secondary metabolism, stress, virulence and nutrient cycling, differences were observed in the functional gene structure of microbial communities and abundance of specific gene families with a threshold between 5 and 10 kg Zn ha−1. However, the richness, diversity and evenness of functional genes did not differ across the zinc gradients in soil. Cluster analysis based on microbial functional subcategories revealed variation in abundances across the gradient of zinc concentrations in soil mainly for virulence, stress, secondary metabolism, and carbon- and phosphorus-cycling-related gene families. A threshold in abundance was observed between 5 and 10 kg Zn ha−1with high abundance of microbial gene families associated with zinc transporter proteins, antioxidant enzymes, exoenzymes associated with infection, secretion proteins, virulence regulatory genes, carbon fixation, and phosphorus utilization in soils supplemented with up to 5 kg Zn ha−1. In these same soils, reduced abundance was observed for gene families associated with methanogenesis, metabolism of halogen and carotenoid, and antiphagocytosis compared with soil supplements with 10 or 20 kg Zn ha−1. The results suggest that changes in microbial functional gene structure and abundance could be used to evaluate the impact of soil management practices on soil microbial communities in sugarcane production fields.