AbstractSharp vertical environmental gradients could differentially affect the structure and function of microbes. Here, the authors provide some of the first insights into the vertical distribution of microbial functions in a polluted urban river using Geochip 5.0 microarray. A total of 89 615 functional genes belonging to 1213 categories are detected in Jinchuan River in China. Genes involved in metal homeostasis, stress response, carbon cycling, antibiotic resistance, and organic contaminant degradation are the primary gene categories detected and comprise 24.63%, 15.20%, 14.17%, 10.59%, and 8.03% of total genes. There are no significant differences in the abundance of functional gene groups at varying depths. Different gene categories showed small fluctuations in their metabolic potentials at varying depths. Functional gene diversity in bottom water and sediment layers is higher than in surface layers. Additionally, functional potentials of antibiotic resistance, organic contaminant degradation, and pigment genes are stronger in water than in sediment, while those of nitrogen cycling and microbial defense increased with depth. The surface sediment layer and surface water layer have the greatest metabolic potential for the carbon cycle and organic pollutant degradation processes, respectively. The middle sediment layer has the highest potential for the nitrogen and phosphorus cycles.