Induction of cardiomyocyte proliferation is a promising option to regenerate the heart. Thus, it is important to elucidate mechanisms that contribute to the cell cycle arrest of mammalian cardiomyocytes. Pericentrin (Pcnt) is a multifunctional scaffold protein that binds to a large variety of centrosomal proteins. Consequently, Pcnt regulates a large number of centrosomal functions such as control of cell cycle progression, mitotic spindle organization and orientation, oriented cell division, and cell fate determination. There are two major alternative splice isoforms of Pcnt, Pcnt B (also known as kendrin) and the shorter Pcnt S, which lacks part of the N-terminal region of Pcnt B. Pcnt B is localized at the centrosome in the vast majority of cell types throughout development. In contrast, Pcnt S is expressed predominantly in the adult heart, skeletal muscle, and testis. Upregulation of Pcnt S is in cardiomyocytes associated with loss of centrosome integrity and cell cycle arrest. After birth, various centrosome proteins, such as Pcnt S, are localized to the nuclear envelope and the paired centrioles, the center of the centrosome, loose cohesion. This process results in split centrioles (> 2 µm apart from each other) and the establishment of a non-centrosomal microtubule organizing center (MTOC) at the nuclear envelope. Besides three studies describing the existence of Pcnt S in the adult heart and skeletal muscle and the localization of Pcnt S at the nuclear envelope in cardiomyocytes, nothing is known about Pcnt S. Considering the role of Pcnt B in cell cycle control and the association of Pcnt S with differentiation and cell cycle arrest in striated muscle cells, we hypothesized that alternative splicing of Pcnt contributes to the establishment of the cell cycle arrest of mammalian cardiomyocytes. Here, we present evidence that alternative splicing of Pcnt contributes to cell cycle control in mammalian cardiomyocytes. We show by antibody staining in combination with siRNA-mediated Pcnt depletion and ectopic expression of tagged Pcnt isoforms that Pcnt B preferentially localizes at the centrosome and Pcnt S at the nuclear envelope.This is further supported by the localization of ectopically expressed FLAG-tagged Pcnt S and Pcnt B in postnatal cardiomyocytes. Analysis of centriole configuration upon Pcnt depletion revealed that Pcnt B but not Pcnt S is required for centriole cohesion. Importantly, ectopic expression of Pcnt S induced centriole splitting in a heterologous system, ARPE-19 cells, and was sufficient to impair DNA synthesis in C2C12 myoblasts. Moreover, Pcnt S depletion enhanced serum-induced cell cycle re-entry in postnatal cardiomyocytes. Analysis of mitosis, binucleation rate, and cell number suggests that Pcnt S depletion promotes progression of postnatal cardiomyocytes through the cell cycle resulting in cell division. Collectively, our data indicate that alternative splicing of Pcnt contributes to the establishment of cardiomyocyte cell cycle arrest shortly after birth.