A significant proportion of breast cancer patients develop bone metastases, but the mechanisms regulating tumor cell dissemination from the primary site to the skeleton remain largely unknown. Using a novel model of spontaneous bone metastasis derived from human ER+ MCF7 cells, molecular profiling revealed increased PREX1 expression in a cell line established from bone-disseminated MCF7 cells (MCF7b), which were more migratory, invasive, and adhesive in vitro compared with parental MCF7 cells, and this phenotype was mediated by PREX1. MCF7b cells grew poorly in the primary tumor site when reinoculated in vivo, suggesting that these cells are primed to grow in the bone, and were enriched in skeletal sites of metastasis over soft tissue sites. Skeletal dissemination from the primary tumor was reversed with PREX1 knockdown, indicating that PREX1 is a key driver of spontaneous dissemination of tumor cells from the primary site to the bone marrow. In breast cancer patients, PREX1 levels are significantly increased in ER+ tumors and associated with invasive disease and distant metastasis. Together, these findings implicate PREX1 in spontaneous bone dissemination and provide a significant advance to the molecular mechanisms by which breast cancer cells disseminate from the primary tumor site to bone.