In preclinical studies, several tumor cell lines have demonstrated an epithelial-to-mesenchymal (EMT)-dependent enhancement in migration when exposed to ionizing radiation at doses of 10 Gy or higher. The goal of this study was to determine whether a lower dose (2.3 Gy) of radiation enhances breast tumor cell migration, and to elucidate the potential contribution of EMT and pro-migratory secreted factors in radiation-induced tumor cell migration. Three human breast cancer cell lines were irradiated and imaged in real-time over 72 h to quantify changes in single cell migration, chemotactic migration and invasion. EMT markers were assessed and conditioned media from irradiated cells was used to determine whether cellular migration was influenced by secreted factors. We observed that a 2.3 Gy dose of radiation did not induce EMT in epithelial-like MCF-7 cells and did not increase the ability of MCF-7 cells or highly motile MDA-MB-231 LM2-4 cells to migrate. In addition, a 2.3 Gy dose significantly increased MDA-MB-231 migration, as detected by single cell tracking and transwell migration assays, but did not increase invasion of MDA-MB-231 cells through reconstituted basement membrane. Cells from all three cell lines migrated further from their point of origin after irradiation, suggesting the cells may be responding to soluble factors produced by other irradiated cells. Consistently, conditioned media derived from 2.3 Gy irradiated MDA-MB-231 cells contained increased levels of several pro-migratory chemokines, and conditioned media from irradiated cells enhanced the migration of nonirradiated MDA-MB-231 cells. These findings indicate that 2.3 Gy dose of radiation is sufficient to increase migration of MDA-MB-231 cells and to alter the single cell migration behavior of three human breast cancer cell lines. Our data suggest the involvement of soluble factors released by 2.3 Gy irradiated cells, and support further in vitro and in vivo studies to identify potential therapeutic targets to prevent tumor cell migration after irradiation.