Abstract: Pancreatic cancer (PC) is one of the most lethal malignancies, and current treatments rarely result in long-term survival. One of the key factors defining its resilience against chemotherapy is the existence of cancer stem cells. c-Myc is a highly amplified oncogene and one of the “most wanted” targets for cancer therapy and maintains tumor stemness, which has so far been an undruggable target. Herein, we describe that a natural cyclopeptide RA-V from Rubia yunnanensis induced ER stress, reduced c-Myc expression and tumor stemness in vitro and vivo. Mechanistically, RA-V induced ER stress-mediated apoptosis through activated the PERK/eIF2α/ATF4/CHOP pathway; RA-V targeted and stabilized the ubiquitin ligase HMG-CoA reductase degradation protein 1 (HRD1), thereby mediating ER-associated degradation (ERAD) of c-Myc protein and decreasing stemness of PC cells. This study provides not only a novel pharmacological strategy to modulate the expression of c-Myc protein, but also new insights into the antitumor mechanism of RA-V.