Cancer stem cells are a subset of cancer cells that initiate the growth of tumors. Low levels of cancer stem cells also exist in established cancer cell lines, and can be enriched in serum-free tumorsphere cultures. Since cancer stem cells have been reported to be resilient to common chemotherapeutic drugs in comparison to regular cancer cells, screening for compounds selectively targeting cancer stem cells may provide an effective therapeutic strategy. We found that 5-azacytidine (5-AzaC) selectively induced anoikis of MCF-7 in suspension cultures with an EC₅₀ of 8.014 µM, and effectively inhibited tumorsphere formation, as well as the migration and matrix metalloproteinases-9 (MMP-9) activity of MCF-7 cells. Furthermore, 5-AzaC and radiation collaboratively inhibited MCF-7 tumorsphere formation at clinically relevant radiation doses. Investigating the underlying mechanism may provide insight into signaling pathways crucial for cancer stem cell survival and pave the way to novel potential therapeutic targets.