The Cre-LoxP system provides a widely used method for studying gene requirements in the mouse as the main mammalian genetic model organism. To define the molecular and cellular mechanisms that underlie cardiovascular development, function and disease, various mouse strains have been engineered that allow Cre-LoxP-mediated gene targeting within specific cell types of the cardiovascular system. Despite the usefulness of this system, evidence is accumulating that Cre activity can have toxic effects in cells, independently of its ability to recombine pairs of engineered LoxP sites in target genes. Here, we have gathered published evidence for Cre toxicity in cells and tissues relevant to cardiovascular biology and provide an overview of mechanisms proposed to underlie Cre toxicity. Based on this knowledge, we propose that each study utilising the Cre-LoxP system to investigate gene function in the cardiovascular system should incorporate appropriate controls to account for Cre toxicity.