Aim: Tropomyosin (Tpm) proteins, encoded by four Tpm genes (Tpm1-4), are associated with the stabilization of the F-actin filaments and play important roles in modulating muscle contraction. So far, little is known about Tpm4 function in embryonic heart development and its involvement in the cardiovascular diseases. In this study, we investigated functions of different isoforms of tpm4 in embryonic heartbeat in zebrafish. Methods and Results: The transgenic zebrafish line, T2EGEZ8, was generated by insertion of a Tol2 transposon gene trap vector, and homozygous mutants (T2EGEZ8(m/m)) of this line showed failure of embryonic heartbeat without other detectable phenotypes. Observation by transmission electron microscopy revealed that the ventricular myocytes of mutant fish contained fewer, disorganized myofibrillar filaments. The transposon genome in T2EGEZ8 fish was found by tail-PCR and RT-PCR to have inserted into the 9th intron of the tpm4 locus, which resulted in production of Tpm4-GFP fusion proteins and loss of normal transcripts tpm4-tv1 and tpm4-tv2. Whole-mount in situ hybridization indicated that tpm4-tv1, encoding a peptide of 284 residues, is specifically expressed in the heart of zebrafish embryos while tpm4-tv2, encoding a peptide of 248 residues, is mainly present in the vasculature but absent in the heart. Knockdown of tpm4-tv1 and tpm4-tv2 within wild-type embryos led to the failure of heartbeat, which could be rescued by coinjection with tpm4-tv1 mRNA but not with tpm4-tv2 mRNA. Conclusion: Tpm4-tv1 is a heart-specific isoform of Tpm4 and is essential for heartbeat in zebrafish embryos.