Aim To study the molecular mechanism of heart high-expressing gene hhole on regulating the transcriptional activity of cardiac hypertrophic signaling molecule ANF. Methods Bioinformatics online software was used to analyse the molecular structure of hhole protein. Several mutants of the recombinant plasmid pCMV-tag2B-hhole were constructed by restriction digestion and site-directed mutagenesis. The mutant and wild-type recombinant plasmids were transfected into HEK293 cell line respectively to determine the transcriptional activity of luciferase reporter gene ANF. Results The bioinformatics analysis reveals that hhole protein contains a binding site of ERK (D-domain) and proline-rich SH3 binding motifs. Four mutants of the recombinant plasmid pCMV-tag2B-hhole have been constructed by restriction digestion and three mutants have been constructed by site-directed mutagenesis. The report assay showed that recombinant plasmids pCMV-tag2B-TD and pCMV-tag2B-hhole could suppress strongly the transcriptional activities of ANF. Conclusion The binding site of ERK-D-domain of hhole protein played a crucial role in inhibiting the transcriptional activities of hypertrophic signal molecule ANF.