p38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G4 structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G-Quadruplex (G4) motifs upstream MAPK12 promoter, forming parallel G4 structures. They exist in competitive binding equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin, binding to different non-contiguous sites at G4 motifs and sequestering either’s occupancy at these motifs to poise MAPK12 transcriptional homeostasis. To under-score the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analyzed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44+/CD24- population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.