This study investigated molecular signals essential to sustain cancer stem cells (CSCs) and assessed their activity in the presence of secreted frizzled-related protein 4 (sFRP4) alone or in combination with chemotherapeutic drugs. SFRP4 is a known Wnt antagonist, and is also pro-apoptotic and anti-angiogenic. Additionally, sFRP4 has been demonstrated to confer chemo-sensitization and improve chemotherapeutic efficacy. CSCs were isolated from breast, prostate, and ovary tumor cell lines, and characterized using tumor-specific markers such as CD44+/CD24-/CD133+. The post-transcription data from CSCs that have undergone combinatorial treatment with sFRP4 and chemotherapeutic drugs suggest downregulation of stemness genes and upregulation of pro-apoptotic markers. The post-translational modification of CSCs demonstrated a chemo-sensitization effect of sFRP4 when used in combination with tumor-specific drugs. SFRP4 in combination with doxorubicin/cisplatin reduced the proliferative capacity of the CSC population in vitro. Wnt/β-catenin signaling is important for proliferation and self-renewal of CSCs in association with human tumorigenesis. The silencing of this signaling pathway by the application of sFRP4 suggests potential for improved in vivo chemo-responses.