—Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (εV1) and translocation activator (ψεRACK [receptors for activated C kinase]) specifically targeting PKCε. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCε signaling. As expected for a PKCε RACK binding peptide, confocal microscopy showed that εV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCε by εV1 at lower expression levels upregulated α–skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, εV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCε translocation with ψεRACK resulted in selectively increased β myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCε signaling in normal postnatal maturational myocardial development and suggest the potential for PKCε activators to stimulate “physiological” cardiomyocyte growth.