The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its interaction with cardiac troponin I, is both a unique expression and heart-enriched gene. To understand the mechanisms of CARK gene expression and regulation, we first cloned the full-length mRNA sequence and mapped the transcription start site of the mouse CARK gene and characterized its promoter regions. Two transcriptional isoforms of the CARK gene were identified in mouse heart tissue. Truncation analysis of the CARK promoter identified a minimal 151 bp region that has strong basal transcription activity. Mutational analysis revealed five conserved cis-acting elements in this 151-bp long minimal promoter. Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter, and CARK transcription level can be down-regulated by MEF2C antisense. Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.