INTRODUCTORY PARAGRAPHCharacterization of the cardiac cellulome—the network of cells that form the heart—is essential for understanding cardiac development and normal organ function, and for formulating precise therapeutic strategies to combat heart disease. Recent studies have challenged assumptions about both the cellular composition1 and functional significance of the cardiac non-myocyte cell pool, with unexpected roles identified for resident fibroblasts2 and immune cell populations3,4. In this study, we characterized single-cell transcriptional profiles of the murine non-myocyte cardiac cellular landscape using single-cell RNA sequencing (scRNA-Seq). Detailed molecular analyses revealed the diversity of the cardiac cellulome and facilitated the development of novel techniques to isolate understudied cardiac cell populations such as mural cells and glia. Our analyses also revealed networks of intercellular communication as well as extensive sexual dimorphism in gene expression in the heart, most notably demonstrated by the upregulation of immune-sensing and pro-inflammatory genes in male cardiac macrophages. This study offers new insights into the structure and function of the mammalian cardiac cellulome and provides an important resource that will stimulate new studies in cardiac cell biology.