Nur77 is one member of the nuclear receptor superfamily. As a transcription factor, Nur77 participates in a variety of biological processes, including T cell development, inflammatory responses, steroid hormone synthesis, and hepatic glucose metabolism. It typically acts via binding to the Nur77 responsive element (NBRE) in the promoter regions of its target genes. In the present study, we identified a novel Nur77-regulated gene, alpha1-antichymotrypsin/SerpinA3, via an approach combining computational prediction and wet-laboratory validations. First, we identified 483 candidate genes via a human genome-wide scan for NBREs in their proximal promoters. Three out of 14 function-associated genes were further identified to be transactivated by Nur77 in luciferase reporter gene assays in HEK 293T cells. The transactivation assay proved that the NBRE (-182 to -175) in the SerpinA3 promoter region is a novel Nur77-dependent functional motif in HEK 293T and HepG2 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that Nur77 physically associates with the SerpinA3 promoter region both in vitro and in vivo. Nur77 overexpression and RNA interference-mediated Nur77 gene knockdown analysis confirmed that SerpinA3 is indeed a novel Nur77-targeted gene. These data may throw light on the function of Nur77 in inflammatory responses and acute-phase reactions as well as the development of Alzheimer's disease.