Similar to the bone marrow, the mammary gland contains a distinct population of Hoechst-effluxing side population cells, mammary gland side population cells (MG-SPs). To better characterize MG-SPs, their microarray gene profiles were compared to the remaining cells, which retain Hoechst dye (mammary gland non-side population cells [MG-NSPs]). For analysis, Gene Ontology (GO) that describes genes in terms of biological processes and Ontology Traverser (OT) that performs enrichment analysis were used. OT showed that MG-SP-specific genes were enriched in the GO categories of cell cycle regulation and checkpoints, multidrug-resistant transporters, organogenesis, and vasculogenesis. The MG-NSP-upregulated genes were enriched in the GO category of cellular organization and biogenesis, which includes basal epithelial markers, p63, smooth muscle actin, myosin, alpha6 integrin, cytokeratin (CK) 14, and luminal markers CK8 and CD24. Additional studies showed that a higher percentage of MG-SPs exist in the G1 phase of the cell cycle compared with the MG-NSPs. G1 cell cycle block of MG-SPs may be explained by higher expression of cell cycle-negative regulatory genes such as transforming growth factor-beta2, insulin-like growth factor binding protein-5, P18(INK4C), and wingless-5a (Wnt-5a). Accordingly, a smaller percentage of MG-SPs expressed nuclear beta-catenin, possibly as a consequence of the higher expression of Wnt-5a. In conclusion, microarray gene profiling suggests that MG-SPs are a lineage-deficient mammary gland subpopulation expressing key genes involved in cell cycle regulation, development, and angiogenesis.