Partial trisomy 2p syndrome includes a spectrum of congenital heart disease (CHD) that is characterized by complex malformations of the outflow and inflow tracts, defects in cardiac septation, heart position, as well as abnormal ventricular development. Lbh (limb-bud and heart) is a novel, highly conserved putative transcriptional regulatory protein, which displays a unique spatiotemporal gene expression pattern during early mouse heart development. Here we show that human LBH maps to chromosome 2p23, a genomic region related to CHD in partial trisomy 2p syndrome. Remarkably, transgenic overexpression of Lbh in mice throughout the embryonic myocardium from a cardiomyocyte-specific promoter of the cardiac ankyrin repeat protein gene (Carp/Ankrd1) models CHD reported in humans with partial trisomy 2p syndrome. The malformations in Carp-Lbh transgenic mice reflect impaired pulmonary outflow tract valvulogenesis, cardiac septation, inflow tract morphogenesis, as well as abnormalities in ventricular cardiomyocyte growth. Furthermore, we demonstrate that overexpression of Lbh in cultured mammalian cells represses the synergistic activity of key cardiac transcription factors, Nkx2.5 and Tbx5, leading to reduced activation of the common target gene, Anf (Nppa). Strikingly, reduced levels of Anf expression were also observed in embryonic day 9.5 Carp-Lbh transgenic mice. Thus, repression of Nkx2.5 and Tbx5-mediated gene expression by deregulated Lbh may account in part for the cardiac anomalies observed in these mice. Our findings implicate LBH as a candidate gene for CHD associated with partial trisomy 2p syndrome and suggest an important role of Lbh in transcriptional control during normal cardiogenesis.