Breast cancer (BC) is the most common type of cancer in women and has a high rate of relapse and death. Notch signaling is crucial for normal breast development and homeostasis. Dysregulation of Notch receptors and ligands has been detected in different BC subtypes and shown to be implicated in tumor development, progression, drug resistance, and recurrence. However, the effects of Notch ligands in various types of BC remain poorly understood. In this study, we investigated the effects of the Notch ligand DLL1 in three different human BC cell lines: MCF-7, BT474, and MDA-MB-231. We showed that DLL1 expression is higher in MCF-7 and BT474 than in MDA-MB-231 cells, and that these cells respond differently to DLL1 downregulation. Functional assays in MCF-7 cells demonstrated that siRNA-mediated DLL1 downregulation reduced colony formation efficiency, migration, proliferation, caused cell cycle arrest at the G1 phase, and induced apoptosis. Gene expression studies revealed that these effects in MCF-7 cells were associated with increased expression of the cell cycle arrest p21 gene and decreased expression of genes that promote cell cycle progression (CDK2, SKP2), and survival (BCL2, BIRC5), unravelling possible mechanisms whereby DLL1 downregulation exerts some of its effects. Moreover, our results demonstrate that treatment with recombinant DLL1 increased MCF-7 cell proliferation and migration, confirming that DLL1 contributes to these processes in this BC cell line. DLL1 downregulation reduced the colony formation efficiency of BT474 cells and decreased the migration and invasion abilities of MDA-MB-231 cells but showed no effects in the proliferation and survival of these cells.