Background— The striated preferentially expressed gene (Speg) generates 4 different isoforms through alternative promoter use and tissue-specific splicing. Depending on the cell type, Speg isoforms may serve as markers of striated or smooth muscle differentiation. Methods and Results— To elucidate function of Speg gene isoforms, we disrupted the Speg gene locus in mice by replacing common exons 8, 9, and 10 with a lacZ gene. β-Galactosidase activity was detected in cardiomyocytes of the developing heart starting at day 11.5 days post coitum (dpc). β-Galactosidase activity in other cell types, including vascular smooth muscle cells, did not begin until 18.5 dpc. In the developing heart, protein expression of only Spegα and Spegβ isoforms was present in cardiomyocytes. Homozygous Speg mutant hearts began to enlarge by 16.5 dpc, and by 18.5 dpc, they demonstrated dilation of right and left atria and ventricles. These cardiac abnormalities in the absence of Speg were associated with a cellular hypertrophic response, myofibril degeneration, and a marked decrease in cardiac function. Moreover, Speg mutant mice exhibited significant neonatal mortality, with increased death occurring by 2 days after birth. Conclusions— These findings demonstrate that mutation of the Speg locus leads to cardiac dysfunction and a phenotype consistent with a dilated cardiomyopathy.