PPP2R2A encodes the B55α regulatory subunit of PP2A, a family of serine/threonine phosphatases that regulate multiple signalling pathways involved in cell proliferation, survival, migration and inflammation. PPP2R2A is a proposed tumour suppressor gene, with recurrent loss detected in a range of cancers, including breast, lung and prostate, however the functional role of B55α loss is not known. Here we show that molecular inhibition of B55α enhances the proliferation, colony formation, anchorage independent growth and in vivo tumour growth of human breast cancer cells. B55α knockdown also enhanced migration and invasion, in association with increased expression of focal adhesion kinase and epithelial-to-mesenchymal transition proteins. Comparative phosphoproteomics identified known and novel proteins regulated by PP2A-B55α, and revealed altered mTOR, RhoA, MAPK, and HIPPO signalling pathways. Of clinical importance, PP2A-B55α knockdown induced resistance to the ER-antagonist, tamoxifen, and the HER2/EGFR inhibitor, lapatinib. However, B55α-knockdown cells were sensitive to a range of PP2A activating drugs. Indeed, PP2A activators sensitized resistant cells to tamoxifen and lapatanib, and inhibited tumour growth and metastases in orthotopic xenografts, suggesting a possible therapeutic option. To further examine the functional role of PP2A-B55α, we generated PP2A-B55α (Ppp2r2a) knockout mice. As with many tumour suppressor genes, constitutive knockout of Ppp2r2a was embryonic lethal, with embryos dying during late development, post 14.5 days p.c. Ppp2r2a-/- embryos displayed neural tube defects (exencephaly and spina bifida), limb defects (non-divergent digits), and impaired epidermal barrier formation. In contrast, mice with mammary specific Pppr2ra deletion were viable, and displayed significantly greater mammary gland branching, supporting a role for PP2A-B55α in mammary gland morphogenesis. Together this work demonstrates the importance of PP2A-B55α in normal development and as a tumour suppressor in breast cancer, and suggests that targeting PP2A activation is a potential therapeutic strategy for poor outcome patients.