BACKGROUND: NXT2 is a new member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes. RESULTS: A zebrafish mutant exhibiting specific heart defects during embryogenesis was generated by animal cloning-mediated retroviral insertions. Molecular analysis indicated that the mutant phenotype was caused by a disruption of NXT2. Whole-mount RNA in situ hybridization showed that NXT2 transcripts were clearly detectable in embryonic heart as well as other tissues. Further analysis revealed that expression level of one form of alternative splicing NXT2 mRNA transcripts was significantly reduced, resulting in deficient myocardial cell differentiation and the malformation of cardiac valve at the atrioventricular boundary. The defects could be reproduced by morpholino anti-sense oligo knockdown of NXT2. CONCLUSIONS: NXT2 has a critical role in maintaining morphogenetic integrity of embryonic heart in vertebrate species.