Following a pathological insult, the adult mammalian heart undergoes hypertrophic growth and remodeling of the extracellular matrix. Although a small subpopulation of cardiomyocytes can reenter the cell cycle following cardiac injury, the myocardium is largely thought to be incapable of significant regeneration. Periostin, an extracellular matrix protein, has recently been proposed to induce reentry of differentiated cardiomyocytes back into the cell cycle and promote meaningful repair following myocardial infarction. Here, we show that although periostin is induced in the heart following injury, it does not stimulate DNA synthesis, mitosis, or cytokinesis of cardiomyocytes in vitro or in vivo. Mice lacking the gene encoding periostin and mice with inducible overexpression of full-length periostin were analyzed at baseline and after myocardial infarction. There was no difference in heart size or a change in cardiomyocyte number in either periostin transgenic or gene-targeted mice at baseline. Quantification of proliferating myocytes in the periinfarct area showed no difference between periostin-overexpressing and -null mice compared with strain-matched controls. In support of these observations, neither overexpression of periostin in cell culture, via an adenoviral vector, nor stimulation with recombinant protein induced DNA synthesis, mitosis, or cytokinesis. Periostin is a regulator of cardiac remodeling and hypertrophy and may be a reasonable pharmacological target to mitigate heart failure, but manipulation of this protein appears to have no obvious effect on myocardial regeneration.