Breast cancer (BC) is women’s most frequent malignant neoplasia and has a high mortality rate. All-trans retinoic acid (RA), a vitamin A-derived pleiotropic signaling molecule, regulates critical genetic programs and shows promise for treating various neoplasias, including BC. However, its use in solid tumors is limited. Previous studies have shown that sensitivity to RA varies among BC cells. We aimed to determine RA’s effects on metastatic processes and characterize the expression profiles of target genes using the Gene Expression Omnibus (GEO) public repository and assess its impact on cell viability in different BC cell lines. We performed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Differential Gene Expression (DGE) analysis, using the GSE103426 database. In-vitro MTT assays and RT-q-PCR experiments were conducted to evaluate the effect of RA on cell viability. Our GO analysis revealed that RA regulates biological processes such as cell migration, motility, epithelial cell differentiation, epithelial-mesenchymal transition, and apoptosis in MDA-MB-231 cells. KEGG analysis indicated that RA downregulates pathways involving differentiation, inflammation, proliferation, angiogenesis, and inhibitory pathways of apoptosis, invasion, migration, and metastasis. Through DGE, we demonstrated that RA modulated the expression of SRC, PTK2, VIM, and CTTN, validated by RT-q-PCR. We confirmed the absence of retinoic acid receptors (RARs) and the reduced ability of RA to inhibit MDA-MB-231 cell viability, indicating resistance to RA. Conversely, the viability of T-47D cells decreased at lower RA doses. In conclusion, despite the absence of RARs, RA affects and modulates mRNA levels in RA-resistant cells, downregulating pathways involved in metastatic processes. These results suggest RA could be significant as a potential therapy for metastatic breast cancers lacking specific treatment options.