CXCR4 overexpression in solid tumors has been strongly associated with poor prognosis and adverse clinical outcome. However, CXCR4 signaling inhibitor drug Plerixafor has shown limited clinical success in cancer treatment. Therefore, CXCR4 signaling may not be the exclusive contributor to its pro-tumorigenic functions. In our continuous effort to understand the chemokine receptor signaling inhibition as cancer therapy, here we unexpectedly discovered that instead of its signaling, intracellular CXCR4 protein augments therapy resistance and pro-tumorigenic functions. Unbiased proteome profiler apoptosis array followed by immunoblot, FACS, real-time PCR and ChIP analyses demonstrate that CXCR4 promotes DR5 downregulation via modulating differential recruitment of transcription factors p53 and YY1 to its promoter. Surprisingly, inhibiting CXCR4 mediated signals failed to block the above phenotype. Irrespective of CXCR4 surface expression, its loss compromised colon tumor growth in vivo. Finally, TCGA data mining and human patient sample data analysis showed CXCR4 and DR5 are inversely regulated in human cancers. Together, we showed evidence for the first time that targeting CXCR4 intracellular protein may be critical to dampen the pro-tumorigenic functions of CXCR4. explain the pro-tumorigenic functions of these axes. In our relentless effort to understand the chemokine receptor signaling inhibition in cancer therapy [15,[19][20][21][22], here we surprisingly discovered that neither CXCR7 nor CXCR4-CXCL12 signaling axis is responsible for therapy resistance and tumorigenic potential, rather CXCR4 protein in the cancer cells plays a very critical role in positively modulating pro-tumorigenic functions such as therapy resistance across multiple solid tumors. Our intricate in vitro and in vivo experiments in multiple cancer settings for CXCR4 gain and loss of function studies demonstrate that CXCR4 protein but not its CXCL12 mediated signals modulate chemotherapy resistance and tumorigenic potential via inversely regulating the expression of DR5. CXCR4-DR5 inverse regulation was also validated in diverse human cell lines and human cancer patient samples, suggesting its possible therapeutic implications.