R-loops are naturally occurring transcriptional intermediates containing RNA/DNA hybrids. Excessive R-loops cause genomic instability, DNA damage, and replication stress. Senataxin-associated exonuclease (San1) is a protein that interacts with Senataxin (SETX), a helicase resolving R-loops. It remains unknown if R-loops-induced DNA damage plays a role in the heart, especially in the proliferative neonatal cardiomyocytes (CMs). San1-/- mice were generated using the CRISPR/Cas9 technique. The newborn San1-/- mice show no overt phenotype, but their hearts were smaller with larger, yet fewer CMs. CM proliferation was impaired with reduced cell cycle-related transcripts and proteins. S9.6 staining revealed that excessive R-loops accumulated in the nucleus of neonatal San1-/- CMs. Increased gamma H2AX staining on newborn and adult heart sections exhibited increased DNA damage. Similarly, San1-/- AC16-cardiomyocytes showed cumulative R-loops and DNA damage, leading to the activation of cell cycle checkpoint kinase ATR and PARP1 hyperactivity, arresting G2/M cell-cycle and CM proliferation. Together, the present study uncovers an essential role of San1 in resolving excessive R-loops that lead to DNA damage and repressing CM proliferation, providing new insights into a novel biological function of San1 in the neonatal heart. San1 may serve as a novel therapeutic target for the treatment of hypoplastic cardiac disorders.