The adult mammalian heart has a limited capacity for regeneration and repair after myocardial infarction. In contrast to adult mice, neonatal mice has remarkable capacity to regenerate injured myocardium until postnatal day 7 (P7). Long non-coding RNAs are shown to have pivotal functions in cardiovascular biology. Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is an evolutionary conserved and highly abundant lncRNA which is reported to be important for endothelial cell proliferation and vessel growth and it is also highly expressed in cardiomyocytes. However, its role in cardiac regeneration is still unclear. Since MALAT1 expression is drastically decreased in adult hearts (P56), and up-upregulated in cardiomyocytes after myocardial infarction (MI) in P1 mice, we assessed its role in neonatal heart regeneration. Malat1^–/– neonates mice showed impaired regeneration capacity measured by the increased fibrosis after MI at postnatal day 3 (P3) when compared to littermate controls (p<0.05, n=9-8). Furthermore, Malat1^-/- mice showed less phosphorylated histone H3 (PHH3; Mitotic marker) positive cardiomyocytes (p<0.05, n=8-10) and less Aurora B⁺ (cytokinesis marker) cardiomyocytes in the border zone (p<0.05, n=5). Interestingly, Malat1^-/- cardiomyocytes showed increased binucleation at P3 indicating incomplete mitosis due to cytokinesis failure (p<0.05, n=5-3). Cardiomyocyte apoptosis, determined by TUNEL staining (p>0.05, n=4), remained unchanged after MI indicating that the failure to regenerate is not due to an increased cell death but involved a failure in cytokinesis. Consistently, GapmeR-mediated silencing of MALAT1 in murine HL-1 cardiomyocyte like cells increased binucleation (p<0.05, n=8). Mechanistically, MALAT1 interacts with hnRNP U (p<0.05, n=3) and tends to control its subcellular localization (p<0.076, n=3), which is involved in the control of cellular proliferation. Taken together, our findings show that genetic deletion or silencing of MALAT1 impaired cardiomyocyte mitosis and increased binucleation, which limits the regeneration of the postnatal heart.