Aspergillus niger has shown great potential for hexavalent chromium [Cr(VI)] bioremediation in wastewater treatment. However, the specific functional genes involved in the Cr(VI) removal pathway in A. niger remain unexplored, limiting our ability to create genetically engineered A. niger strains with enhanced bioremediation capabilities. In this study, we successfully identified two critical genes in A. niger responsible for chromate removal: chrR, a putative gene encoding chromate reductase, and chrP, a putative gene encoding chromate permease. Our findings revealed that deletion of either chrR or chrP in A. niger significantly impaired Cr(VI) removal. Conversely, overexpressing chrR or chrP increased Cr(VI) removal rates by 34.0% and 29.7%, respectively, compared to the parental strain. Additionally, our study showed that sodA, one of the three cytoplasmic superoxide dismutase genes in A. niger, plays a pivotal role in detoxifying reactive oxygen species (ROS) stress induced by Cr(IV). Furthermore, by cooverexpressing chrR, chrP, and sodA to simultaneously enhance the Cr(VI) uptake, reduction, and ROS detoxification pathway, we successfully engineered an A. niger strain that exhibited a remarkable 92% improvement in Cr(VI) removal compared to the wildtype strain. This achievement highlights the significant potential of genetic engineering in addressing Cr(VI) pollution through bioremediation.