The diversity and biogeography of functional genes at a regional scale remains unclear. We hypothesize that plant community composition, nutrient availability, and spatial distance are driving factors for the functional gene diversity of soil microorganisms at a regional scale. We also hypothesize that different functional genes have different responses to environment. Using functional gene arrays (GeoChip 3.0), we examined microbial diversity, and influencing factors in Ultisols in 11 natural secondary forest plots in southern China which contained 4073 functional genes averagely. Carbon and nitrogen cycle genes were more variable than genes involved in phosphorus utilization, organic remediation, and metal resistance, in similar trends with soil substrate availability. Genes involved in nitrification being more variable than other nitrogen cycling genes; phytase genes being more variable than polyphosphate kinase genes (ppk) and exopolyphosphatase genes (ppx); exoglucanase and endoglucanase genes being more variable than cellobiase genes; lignin peroxidase genes (lip) being more variable than manganese peroxidase genes (mnp). Approximately 41% of the variation in gene composition could be explained by fine root surface area, available nitrogen, and the largest spatial scale factor, with the former two environmental factors over influenced spatial distance. Both environmental factors (represented by nutrient availability) and spatial distance influenced microbial functional gene composition, while the influence of spatial distance worked through environmental factors in natural secondary forest in Ultisols within approximately 500 km, providing insights into microbial biogeography from a functional gene perspective.