Actinomycin D (ActD) is a natural antibiotic that inhibits the transcription of genes by interacting with a GC-rich duplex, a single-stranded or hairpin form of DNA, and then interfering with the action of RNA polymerase. In this study, we identified a novel molecular mechanism of anticancer activity of ActD as an oncogenic c-Myc promoter G-quadruplex binder. ActD selectively inhibits the elongation of oligonucleotides containing c-Myc promoter G-quadruplex sequence in PCR-stop assays. UV-vis spectroscopic and circular dichroism studies suggest that ActD interacts with c-Myc promoter G-quadruplex via a surface end stacking interaction, inducing a mixed-type conformation of the G-quadruplex. ActD selectively inhibits the cellular growth and synthesis of c-Myc mRNA in Ramos cells having the NHEIII(1) region in the translocated c-Myc gene. In addition, the results of promoter assays using two kinds of NHEIII(1) region mutants and wild-type constructs strongly support the idea that binding of ActD with G-quadruplex formed in the promoter region results in the reporter gene being turned off. Our study reveals a novel mechanism underlying the anticancer activity of ActD, whereby ActD interacts with oncogenic promoter G-quadruplex DNA to repress gene expression.