Annexin 1 (ANXA1), the first characterized member of the annexin superfamily, is known to bind or annex to cellular membranes in a calcium-dependent manner. Besides mediating inflammation, ANXA1 has also been reported to be involved in important physiopathological implications including cell proliferation, differentiation, apoptosis, cancer, and metastasis. However, with controversies in ANXA1 expression in breast carcinomas, its role in breast cancer initiation and progression remains unclear. To elucidate how ANXA1 plays a role in breast cancer initiation, we performed stable isotope labeling of amino acids in cell culture analysis on normal mammary gland epithelial cells from ANXA1-heterozygous (ANXA1(+/-)) and ANXA1-null (ANXA1(-/-)) mice. Among over 4000 quantified proteins, we observed 214 up-regulated and 169 down-regulated with ANXA1(-/-). Bioinformatics analysis of the down-regulated proteins revealed that ANXA1 is potentially implicated in DNA damage response, whereas the analysis of up-regulated proteins showed the possible roles of ANXA1 in cell adhesion and migration pathways. These observations were supported by relevant functional assays. The assays for DNA damage response demonstrated an accumulation of more DNA damage with slower recovery on heat stress and an impaired oxidative damage response in ANXA1(-/-) cells in comparison with ANXA1(+/-) cells. Overexpressing Yes-associated protein 1 or Yap1, the most down-regulated protein in DNA damage response pathway cluster, rescued the proliferative response in ANXA1(-/-) cells exposed to oxidative damage. Both migration and wound healing assays showed that ANXA1(+/-) cells possess higher motility with better wound closure capability than ANXA1(-/-) cells. Knocking down of β-parvin, the protein with the highest fold change in the cell adhesion protein cluster, indicated an increased cell migration in ANXA1(-/-) cells. Altogether our quantitative proteomics study on ANXA1 suggests that ANXA1 plays a protective role in DNA damage and modulates cell adhesion and motility, indicating its potential role in cancer initiation as well as progression in breast carcinoma.