The serum response factor (SRF) is a transcriptional regulator required for mesodermal development, including heart formation and function. Previous studies have described the role of SRF in controlling expression of structural genes involved in conferring the myogenic phenotype. Recent studies by us and others have demonstrated embryonic lethal cardiovascular phenotypes in SRF-null animals, but have not directly addressed the mechanistic role of SRF in controlling broad regulatory programs in cardiac cells. In this study, we used a loss-of-function approach to delineate the role of SRF in cardiomyocyte gene expression and function. In SRF-null neonatal cardiomyocytes, we observed severe defects in the contractile apparatus, including Z-disc and stress fiber formation, as well as mislocalization and/or attenuation of sarcomeric proteins. Consistent with this, gene array and reverse transcription-PCR analyses showed down-regulation of genes encoding key cardiac transcriptional regulatory factors and proteins required for the maintenance of sarcomeric structure, function, and regulation. Chromatin immunoprecipitation analysis revealed that at least a subset of these proteins are likely regulated directly by SRF. The results presented here indicate that SRF is an essential coordinator of cardiomyocyte function due to its ability to regulate expression of numerous genes (some previously identified and at least 28 targets newly identified in this study) that are involved in multiple and disparate levels of sarcomeric function and assembly.