Naphthazarin (Naph, DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is one of the naturally available 1,4-naphthoquinone derivatives that are well-known for their anti-inflammatory, antioxidant, antibacterial and antitumor cytotoxic effects in cancer cells. Herein, we investigated whether Naph has effects on cell cycle arrest and apoptosis in MCF-7 human breast cancer cells exposed to ionizing radiation (IR). Naph reduced the MCF-7 cell viability in a dose-dependent manner. We also found that Naph and/or IR increased the p53-dependent p21 (CIP/WAF1) promoter activity. Noteworthy, our ChIP assay results showed that Naph and IR combined treatment activated the p21 promoter via inhibition of binding of multi-domain proteins, DNMT1, UHRF1 and HDAC1. Apoptosis and cell cycle analyses demonstrated that Naph and IR combined treatment induced cell cycle arrest and apoptosis in MCF-7 cells. Herein, we showed that Naph treatment enhances IR-induced cell cycle arrest and death in MCF-7 human breast cancer cells through the p53-dependent p21 activation mechanism. These results suggest that Naph might sensitize breast cancer cells to radiotherapy by enhancing the p53-p21 mechanism activity.