Cardia bifida is an anomaly of the embryonic heart in which the bilateral myocardial rudiments fail to travel to the midline, resulting in the formation of two separate hearts in lateral positions. In zebrafish, eight loci responsible for the cardia bifida phenotype were identified in the large-scale genetic screen. Wortmannin has been reported to be a highly selective inhibitor of phosphoinositide 3-kinase and myosin light chain kinase activity. We provide the first evidence that wortmannin treatment of zebrafish embryos can induce cardia bifida in a dose-dependent manner and that wortmannin alters cardiac development between 6 and 16h post-fertilization. In addition, we demonstrate that wortmannin induces zebrafish cardia bifida through a mechanism independent of phosphoinositide 3-kinase and myosin light chain kinase. Our findings may provide new insights into the cardiomyocyte function and disfunction.