Cyclooxygenase (COX) encodes a rate-limiting enzyme in the biosynthesis of prostanoids. Although COX-1 is constitutively expressed in many tissues, we found that glucocorticoids cause elevated expression of COX-1 gene in cardiomyocytes. Corticosterone (CT) at physiologically relevant doses (0.05 – 1 µM) induces transcriptional activation of COX-1 gene as shown by nuclear run-on and promoter reporter assays. An antagonist of glucocorticoid receptor (GR), mifepristone (MF), prevented CT from inducing COX-1. COX-1 gene promoter deletion and mutation studies indicate a role of Sp transcription factors in CT induced COX-1 gene. Electrophoretic mobility shift assays or chromatin immunoprecipitation assays suggest that GR and Sp3 transcription factor bind to the promoter of COX-1 gene. Co-immunoprecipitation assays found an association of GR with Sp3. Silencing Sp3 protein with small interfering RNA suppressed CT-induced COX-1 promoter activation. Our data suggest that activated GR interacts with Sp3 transcription factor in binding to COX-1 promoter to enhance COX-1 gene expression in cardiomyocytes.