Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that Isoproterenol (ISO) induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a non-selective β-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. QPCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO responsive element was mapped to the proximal -100bp sequence of the CDK1 promoter region using various 5' flanking sequence deletion constructs. Sequence analysis of the -100bp CDK1 minimal promoter region revealed two putative NF-Y binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of -100bp CDK1 promoter construct but not the ISO induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site directed mutagenesis of the distal NF-Y binding site in the -100bp CDK1 promoter region completely abolished both basal and ISO induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.